Why do <Emphasis Type="Italic">Manduca sexta</Emphasis> feed from white flowers? Innate and learnt colour preferences in a hawkmoth

Flower colour is an important signal used by flowering plants to attract pollinators. Many anthophilous insects have an innate colour preference that is displayed during their first foraging bouts and which could help them locate their first nectar reward. Nevertheless, learning capabilities allow insects to switch their colour preferences with experience and thus, to track variation in floral nectar availability. Manduca sexta, a crepuscular hawkmoth widely studied as a model system for sensory physiology and behaviour, visits mostly white, night-blooming flowers lacking UV reflectance throughout its range in the Americas. Nevertheless, the spectral sensitivity of the feeding behaviour of naïve moths shows a narrow peak around 450 nm wavelengths, suggesting an innate preference for the colour blue. Under more natural conditions (i.e. broader wavelength reflectance) than in previous studies, we used dual choice experiments with blue- and white-coloured feeders to investigate the innate preference of naïve moths and trained different groups to each colour to evaluate their learning capabilities. We confirmed the innate preference of M. sexta for blue and found that these moths were able to switch colour preferences after training experience. These results unequivocally demonstrate that M. sexta moths innately prefer blue when presented against white flower models and offer novel experimental evidence supporting the hypothesis that learning capabilities could be involved in their foraging preferences, including their widely observed attraction to white flowers in nature.     

A comparative analysis of colour preferences in temperate and tropical social bees

The spontaneous occurrence of colour preferences without learning has been demonstrated in several insect species; however, the underlying mechanisms are still not understood. Here, we use a comparative approach to investigate spontaneous and learned colour preferences in foraging bees of two tropical and one temperate species. We hypothesised that tropical bees utilise different sets of plants and therefore might differ in their spontaneous colour preferences. We tested colour-naive bees and foragers from colonies that had been enclosed in large flight cages for a long time. Bees were shortly trained with triplets of neutral, UV-grey stimuli placed randomly at eight locations on a black training disk to induce foraging motivation. During unrewarded tests, the bees’ responses to eight colours were video-recorded. Bees explored all colours and displayed an overall preference for colours dominated by long or short wavelengths, rather than a single colour stimulus. Naive Apis cerana and Bombus terrestris showed similar choices. Both inspected long-wavelength stimuli more than short-wavelength stimuli, whilst responses of the tropical stingless bee Tetragonula iridipennis differed, suggesting that resource partitioning could be a determinant of spontaneous colour preferences. Reward on an unsaturated yellow colour shifted the bees’ preference curves as predicted, which is in line with previous findings that brief colour experience overrides the expression of spontaneous preferences. We conclude that rather than determining foraging behaviour in inflexible ways, spontaneous colour preferences vary depending on experimental settings and reflect potential biases in mechanisms of learning and decision-making in pollinating insects.     

Pollen foraging: learning a complex motor skill by bumblebees (<Emphasis Type="Italic">Bombus terrestris</Emphasis>)

To investigate how bumblebees (Bombus terrestris) learn the complex motor skills involved in pollen foraging, we observed naïve workers foraging on arrays of nectarless poppy flowers (Papaver rhoeas) in a greenhouse. Foraging skills were quantified by measuring the pollen load collected during each foraging bout and relating this to the number of flowers visited and bout duration on two consecutive days. The pollen standing crop (PSC) in each flower decreased drastically from 0530 to 0900 hours. Therefore, we related foraging performance to the changing levels of pollen available (per flower) and found that collection rate increased over the course of four consecutive foraging bouts (comprising between 277 and 354 individual flower visits), suggesting that learning to forage for pollen represents a substantial time investment for individual foragers. The pollen collection rate and size of pollen loads collected at the start of day 2 were markedly lower than at the end of day 1, suggesting that components of pollen foraging behaviour could be subject to imperfect overnight retention. Our results suggest that learning the necessary motor skills to collect pollen effectively from morphologically simple flowers takes three times as many visits as learning how to handle the most morphologically complex flowers to extract nectar, potentially explaining why bees are more specialised in their choice of pollen flowers.     

Time management and nectar flow: flower handling and suction feeding in long-proboscid flies (Nemestrinidae: Prosoeca)

A well-developed suction pump in the head represents an important adaptation for nectar-feeding insects, such as Hymenoptera, Lepidoptera and Diptera. This pumping organ creates a pressure gradient along the proboscis, which is responsible for nectar uptake. The extremely elongated proboscis of the genus Prosoeca (Nemestrinidae) evolved as an adaptation to feeding from long, tubular flowers. According to the functional constraint hypothesis, nectar uptake through a disproportionately elongated, straw-like proboscis increases flower handling time and consequently lowers the energy intake rate. Due to the conspicuous length variation of the proboscis of Prosoeca, individuals with longer proboscides are hypothesised to have longer handling times. To test this hypothesis, we used field video analyses of flower-visiting behaviour, detailed examinations of the suction pump morphology and correlations of proboscis length with body length and suction pump dimensions. Using a biomechanical framework described for nectar-feeding Lepidoptera in relation to proboscis length and suction pump musculature, we describe and contrast the system in long-proboscid flies. Flies with longer proboscides spent significantly more time drinking from flowers. In addition, proboscis length and body length showed a positive allometric relationship. Furthermore, adaptations of the suction pump included an allometric relationship between proboscis length and suction pump muscle volume and a combination of two pumping organs. Overall, the study gives detailed insight into the adaptations required for long-proboscid nectar feeding, and comparisons with other nectar-sucking insects allow further considerations of the evolution of the suction pump in insects with sucking mouthparts.     

Flower-visiting behavior of male bees is triggered by nectar-feeding insects

Bees are important pollinators for many flowering plants. Female bees are thought to be more effective pollinators than male bees because they carry much more pollen than males. Males of some solitary bee species are known to patrol near flowers that females visit. Because patrolling males visit flowers to mate or defend their territories, they may function as pollinators. However, the significance of patrolling males to pollination has not been studied. We studied males of a solitary bee, Heriades fulvohispidus (Megachilidae), patrolling near flowers and visiting flowers that attracted nectar-feeding insects, including conspecifics, on the Ogasawara (Bonin) Islands. To test the hypothesis that patrolling male bees may function as pollen vectors, we compared the frequency of visits by H. fulvohispidus to flowers of an endemic plant, Schima mertensiana (Theaceae); comparisons were made among flowers with a dead H. fulvohispidus, a dead beetle, a piece of plastic, and nothing (control flowers). Patrolling H. fulvohispidus more frequently visited flowers with a dead conspecific, a dead beetle, or a piece of plastic than the control flowers. Our experiment demonstrates that nectar-feeding insects (including conspecifics and other insects) enhance the flower-visiting frequency of patrolling H. fulvohispidus males on S. mertensiana flowers. Furthermore, we observed S. mertensiana pollen on patrolling males as well as females, suggesting that male bees may also function as pollen vectors.     

Large pollen loads of a South African asclepiad do not interfere with the foraging behaviour or efficiency of pollinating honey bees

The pollen of asclepiads (Asclepiadoideae, Apocynaceae) and most orchids (Orchidaceae) are packaged as large aggregations known as pollinaria that are removed as entire units by pollinators. In some instances, individual pollinators may accumulate large loads of these pollinaria. We found that the primary pollinator of Cynanchum ellipticum (Apocynaceae—Asclepiadoideae), the honey bee Apis mellifera, accumulate very large agglomerations of pollinaria on their mouthparts when foraging on this species. We tested whether large pollinarium loads negatively affected the foraging behaviour and foraging efficiency of honey bees by slowing foraging speeds or causing honey bees to visit fewer flowers, and found no evidence to suggest that large pollinarium loads altered foraging behaviour. C. ellipticum displayed consistently high levels of pollination success and pollen transfer efficiency (PTE). This may be a consequence of efficiently loading large numbers of pollinaria onto pollinators even when primary points of attachment on pollinators are already occupied and doing so in a manner that does not impact the foraging behaviour of pollinating insects.     

Floral odor learning within the hive affects honeybees’ foraging decisions

Honeybees learn odor cues quickly and efficiently when visiting rewarding flowers. Memorization of these cues facilitates the localization and recognition of food sources during foraging flights. Bees can also use information gained inside the hive during social interactions with successful foragers. An important information cue that can be learned during these interactions is food odor. However, little is known about how floral odors learned in the hive affect later decisions of foragers in the field. We studied the effect of food scent on foraging preferences when this learning is acquired directly inside the hive. By using in-hive feeders that were removed 24 h before the test, we showed that foragers use the odor information acquired during a 3-day stimulation period with a scented solution during a food-choice situation outside the nest. This bias in food preference is maintained even 24 h after the replacement of all the hive combs. Thus, without being previously collected outside by foragers, food odors learned within the hive can be used during short-range foraging flights. Moreover, correct landings at a dual-choice device after replacing the storing combs suggests that long-term memories formed within the colony can be retrieved while bees search for food in the field.     

Folivory versus florivory—adaptiveness of flower feeding

The distribution of resources and defence is heterogeneous within plants. Specialist insects may prefer tissue with high concentrations of the plant’s characteristic defence compounds. Most herbivorous butterfly or sawfly larvae are considered to be folivores, so also the turnip sawfly Athalia rosae (Hymenoptera: Tenthredinidae), a specialist herbivore on Brassicaceae. We investigated which tissue larvae choose to feed upon and how they perform on flowers, young or old leaves of Sinapis alba. Furthermore, constitutive and inducible levels of glucosinolates and myrosinases were investigated and nutrients analysed. Larvae moved from leaves to flowers for feeding from the third larval instar on. Flowers were not actively chosen, but larvae moved upwards on the plant, regardless of how plants were orientated (upright or inverted). Flower-feeding larvae were heavier and developed faster than larvae feeding on young leaves, and adults laid more eggs. Old leaves as food source resulted in the lowest growth rates. Flowers contained three and ten times higher myrosinase activities than young and old leaves, respectively, whereas glucosinolate concentrations and nitrogen levels of flowers and young leaves were comparable. Glucosinolate concentrations of old leaves were very low. Changes in tissue chemistry caused by larval feeding were tissue specific. Defence levels did not change in flowers and old leaves after A. rosae feeding in contrast to young leaves. The high insect performance on flowers cannot be explained by differences in chemical defence. Instead, the lack of mechanical defence (trichomes) is probably responsible. Movement to the flowers and folivory is overall highly adaptive for this sawfly species.     

How floral odours are learned inside the bumblebee (Bombus terrestris) nest

Recruitment in social insects often involves not only inducing nestmates to leave the nest, but also communicating crucial information about finding profitable food sources. Although bumblebees transmit chemosensory information (floral scent), the transmission mechanism is unknown as mouth-to-mouth fluid transfer (as in honeybees) does not occur. Because recruiting bumblebees release a pheromone in the nest that triggers foraging in previously inactive workers, we tested whether this pheromone helps workers learn currently rewarding floral odours, as found in food social learning in rats. We exposed colonies to artificial recruitment pheromone, paired with anise scent. The pheromone did not facilitate learning of floral scent. However, we found that releasing floral scent in the air of the colony was sufficient to trigger learning and that learning performance was improved when the chemosensory cue was provided in the nectar in honeypots; probably because it guarantees a tighter link between scent and reward, and possibly because gustatory cues are involved in addition to olfaction. Scent learning was maximal when anise-scented nectar was brought into the nest by demonstrator foragers, suggesting that previously unidentified cues provided by successful foragers play an important role in nestmates learning new floral odours.     

Different patterns of oviposition learning in two closely related ectoparasitoid wasps with contrasting reproductive strategies

Many parasitoid wasps learn host-associated cues and use them in subsequent host-searching behavior. This associative learning, namely “oviposition learning,” has been investigated in many studies. However, few studies have compared multiple species, and no comparative study has previously been conducted on ectoparasitoid species. We compared the effects of oviposition learning on host preference and offspring sex ratio in two closely related ectoparasitoid wasps with contrasting reproductive strategies, Anisopteromalus calandrae (r-strategist) and its sibling species (K-strategist). Using two bruchine hosts, Callosobruchus chinensis and Callosobruchus maculatus larvae infesting the cowpea Vigna unguiculata, oviposition choice experiments were performed at high and low host densities. In both species, no conspicuous effect on the offspring sex ratio was detected, but effects on host preference were found to differ between the species. In A. calandrae, the effects were detected only at high host density, suggesting that oviposition learning plays a role in host discrimination from a short distance but not from a long distance. In the sibling species, those effects were not detected in any of the cases, suggesting the absence of oviposition learning. These results are compatible with those of previous comparative studies of endoparasitoid wasps in that few lifetime oviposition experiences and/or low reward per foraging decision result in low or absent oviposition learning ability. This finding may indicate that ecological traits contributing to learning ability are similar between endoparasitoid and ectoparasitoid wasps. Thus, our species comparison of ectoparasitoids provides another model system for investigating learning and memory dynamics in parasitoid wasps.     

Floral colour change in <Emphasis Type="Italic">Byrsonima variabilis</Emphasis> (Malpighiaceae) as a visual cue for pollen but not oil foraging by oil-collecting bees

Pollinators search for multiple flora resources throughout their life cycle. Most studies, however, only assess how bees discriminate floral cues in the context of nectar foraging. In the present study, we sought to elucidate whether oil-collecting bees discriminate flowers of Byrsonima variabilis (Malpighiaceae) with petals of different colours when foraging for pollen or oil. As the colour of the standard petal changes during anthesis, we characterised the spectral reflectance patterns of flowers throughout anthesis and modelled chromatic perceptual space to determine how these colour patterns are perceived by bees. Through the quantification of flower pollen in the different phases, we found that the colour of the standard petal is an honest cue of the presence of pollen. Centridine bees preferentially visited flowers with a yellow (bee’s green) colour when searching for pollen, but indiscriminately visited flowers with different petal colours when searching for floral oil. We suggest that standard petals, in the species studied and others of the genus, like nectar guides, act as pollen guides, which oil-collecting females use to detect pollen-rich flowers. Moreover, they use different floral clues during foraging for different resources in the same host plant.     

Procurement of exogenous ammonia by the swallowtail butterfly, Papilio polytes, for protein biosynthesis and sperm production

How to acquire sufficient quantity of nitrogen is a pivotal issue for herbivores, particularly for lepidopterans (butterflies and moths) of which diet quality greatly differs among their life stages. Male Lepidoptera often feed from mud puddles, dung, and carrion, a behavior known as puddling, which is thought to be supplementary feeding targeted chiefly at sodium. During copulation, males transfer a spermatophore to females that contains, besides sperm, nutrients (nuptial gifts) rich in sodium, proteins, and amino acids. However, it is still poorly understood how adults, mostly nectarivores, extract nitrogen from the environment. We examined the availability of two ubiquitous inorganic nitrogenous ions in nature, viz. ammonium (or ammonia) and nitrate ions, as nutrients in a butterfly, and show that exogenous ammonia ingested by adult males of the swallowtail, Papilio polytes, can serve as a resource for protein biosynthesis. Feeding experiments with ¹⁵N-labeled ammonium chloride revealed that nitrogen was incorporated into eupyrene spermatozoa, seminal protein, and thoracic muscle. Ammonia uptake by males significantly increased the number of eupyrene sperms in the reproductive tract tissues. The females also had the capacity to assimilate ammonia into egg protein. Consequently, it is evident that acquired ammonia is utilized for the replenishment of proteins allocable for reproduction and somatic maintenance. The active exploitation of exogenous ammonia as a nutrient by a butterfly would foster better understanding of the foraging and reproductive strategies in insects.     

Flower Constancy, Insect Psychology, and Plant Evolution

 Individuals of some species of pollinating insects tend to restrict their visits to only a few of the available plant species, in the process bypassing valuable food sources. The question of why this flower constancy exists is a rich and important one with implications for the organization of natural communities of plants, floral evolution, and our understanding of the learning processes involved in finding food. Some scientists have assumed that flower constancy is adaptive per se. Others argued that constancy occurs because memory capacity for floral features in insects is limited, but attempts to identify the limitations often remained rather simplistic. We elucidate now different sensory and motor memories from natural foraging tasks are stored and retrieved, using concepts from modern learning science and visual search, and conclude that flower constancy is likely to have multiple causes. Possible constraints favoring constancy are interference sensitivity of short-term memory, and temporal limitations on retrieving information from long-term memory as rapidly as from short-term memory, but further empirical evidence is needed to substantiate these possibilities. In addition, retrieving memories may be slower and more prone to errors when there are several options than when an insect copes with only a single task. In addition to memory limitations, we also point out alternative explanations for flower constancy. We then consider the way in which floral parameters, such as interplant distances, nectar rewards, flower morphology, and floral color (as seen through bees' eyes) affect constancy. Finally, we discuss the implications of pollinator constancy for plant evolution. To date there is no evidence that flowers have diverged to favor constancy, although the appropriate tests may not have yet been conducted. However, there is good evidence against the notion that pollinator constancy is involved in speciation or maintenance of plant species integrity.     

Sexual differences in food preferences in the white stork: an experimental study

Sex differences in the foraging ecology of monomorphic species are poorly understood, due to problems with gender identification in field studies. In the current study, we used experimental conditions to investigate the food preferences of the white stork Ciconia ciconia, an opportunistic species in terms of food, but characterised by a low level of sexual dimorphism. During a 10-day experiment, 29 individuals (20 females and 9 males) were studied by means of a ‘cafeteria test’ in which the storks’ diet consisted of mammals, birds, fish, amphibians, insects and earthworms. The storks preferred food characterised by high calorific and protein values such as mammals, birds and fish. Sexes differed strongly in their preferences; males preferred mammals, whereas females preferred birds. Moreover, females consumed insects and earthworms less often than males. Interestingly, males spent significantly less time foraging than females. We have demonstrated that the white stork exhibits clear sexual differences in food preferences which are mostly attributable to differences in parental duties, physiology and anatomy.     

The effect of octopamine on behavioral responses of free-foraging bumblebees to a change in food source profitability

The invertebrate neuromodulator octopamine is known to be involved in bees' associative learning, enhancing the responsiveness of a bee to a conditioned stimulus. In this study, we tested the effect of octopamine on the choice behavior of free-flying bumblebees using a two-phase experiment in an array of artificial flowers. During the first phase of the experiment, the bee was allowed to collect octopamine-laden sugar water from two types of equally rewarding flowers (yellow versus blue). In the second phase, one type of flower was set to be unrewarding. The behavior of the bee (proportion of visits to the unrewarding flowers) over the two phases was fitted to a sigmoid regression model. Our results show that octopamine had no significant effect on the bees' equilibrium choice or on the overall rate of the behavioral change in response to the change in reward. Rather, octopamine significantly affected the time interval between the change in reward status and the initiation of behavioral change in the bee.     

Nutrient compensatory foraging in a free-living social insect

The geometric framework model predicts that animal foraging decisions are influenced by their dietary history, with animals targeting a combination of essential nutrients through compensatory foraging. We provide experimental confirmation of nutrient-specific compensatory foraging in a natural, free-living population of social insects by supplementing their diet with sources of protein- or carbohydrate-rich food. Colonies of the ant Iridomyrmex suchieri were provided with feeders containing food rich in either carbohydrate or protein for 6 days, and were then provided with a feeder containing the same or different diet. The patterns of recruitment were consistent with the geometric framework: while feeders with a carbohydrate diet typically attracted more workers than did feeders with protein diet, the difference in recruitment between the two nutrients was smaller if the colonies had had prior access to carbohydrate than protein. Further, fewer ants visited feeders if the colony had had prior access to protein than to carbohydrates, suggesting that the larvae play a role in worker foraging behaviour.     

Social learning of an associative foraging task in zebrafish

The zebrafish (Danio rerio) is increasingly becoming an important model species for studies on the genetic and neural mechanisms controlling behaviour and cognition. Here, we utilized a conditioned place preference (CPP) paradigm to study social learning in zebrafish. We tested whether social interactions with conditioned demonstrators enhance the ability of focal naïve individuals to learn an associative foraging task. We found that the presence of conditioned demonstrators improved focal fish foraging behaviour through the process of social transmission, whereas the presence of inexperienced demonstrators interfered with the learning of the control focal fish. Our results indicate that zebrafish use social learning for finding food and that this CPP paradigm is an efficient assay to study social learning and memory in zebrafish.     

Chemically mediated group formation in soil-dwelling larvae and pupae of the beetle Trypoxylus dichotomus

Many insects form groups through interactions among individuals, and these are often mediated by chemical, acoustic, or visual cues and signals. In spite of the diversity of soil-dwelling insects, their aggregation behaviour has not been examined as extensively as that of aboveground species. We investigated the aggregation mechanisms of larvae of the Japanese rhinoceros beetle Trypoxylus dichotomus, which live in groups in humus soil. In two-choice laboratory tests, 2nd- and 3rd-instar larvae gathered at conspecific larvae irrespective of the kinship. The ablation of maxillae, which bear chemosensilla, abolished aggregation behaviour. Intact larvae also exhibited aggregation behaviour towards a larval homogenate. These results suggest that larval aggregation is mediated by chemical cues. We also demonstrated that the mature larvae of T. dichotomus built their pupal cells close to a mesh bag containing a conspecific pupal cell, which indicated that larvae utilize chemical cues emanating from these cells to select the pupation site. Thus, the larvae of T. dichotomus may use chemical cues from the conspecifics in two different contexts, i.e. larval aggregation and pupation site selection. Using conspecific cues, larvae may be able to choose suitable locations for foraging or building pupal cells. The results of the present study highlight the importance of chemical information in belowground ecology.     

Comparison of pollination and defensive buzzes in bumblebees indicates species-specific and context-dependent vibrations

Bees produce vibrations in many contexts, including for defense and while foraging. Buzz pollination is a unique foraging behavior in which bees vibrate the anthers of flowers to eject pollen which is then collected and used as food. The relationships between buzzing properties and pollen release are well understood, but it is less clear to what extent buzzing vibrations vary among species, even though such information is crucial to understanding the functional relationships between bees and buzz-pollinated plants. Our goals in this study were (1) to examine whether pollination buzzes differ from those produced during defense, (2) to evaluate the similarity of buzzes between different species of bumblebees (Bombus spp.), and (3) to determine if body size affects the expression of buzzing properties. We found that relative peak amplitude, peak frequency, and duration were significantly different between species, but only relative peak amplitude differed between pollination and defensive buzzes. There were significant interactions between species and buzz type for peak frequency and duration, revealing that species differed in their patterns of expression in these buzz properties depending on the context. The only parameter affected by body size was duration, with larger bees producing shorter buzzes. Our findings suggest that although pollination and defensive buzzes differ in some properties, variability in buzz structure also exhibits a marked species-specific component. Species differences in pollination buzzes may have important implications for foraging preferences in bumblebees, especially if bees select flowers best matched to release pollen for their specific buzzing characteristics.     

The use of heterospecific scent marks by the sweat bee <Emphasis Type="Italic">Halictus aerarius</Emphasis>

To forage effectively amongst flowers, some bee species utilize olfactory cues left by previous visitors in addition to direct assessment of visual cues to identify rewarding flowers. This ability can be more advantageous if the bees can recognize and use scent marks left by heterospecifics, not just marks left by members of their own species. We conducted field experiments to investigate whether the sweat bee Halictus aerarius avoids visiting flowers of trailing water willow Justicia procumbens emptied by other bee species. We found that H. aerarius rejected the flowers visited by both heterospecifics and conspecifics. They also rejected visited flowers artificially replenished with nectar. Our results demonstrate that social bees outside the Apidae can detect marks left on flowers by heterospecifics but that (on this plant species) they are unable to discriminate against flowers by directly detecting nectar volume. H. aerarius exhibited different rejection rates according to the identity of the previous bee species. We suggest that the frequency of rejection responses may depend on the amount of chemical substances left by the previous bee. In general, the use of scent marks left by previous visitors is almost certainly advantageous, enabling foragers to avoid flowers with depleted nectar levels and thereby improving their foraging efficiency.