Correction: Associative Mechanisms Allow for Social Learning and Cultural Transmission of String Pulling in an Insect.

[This corrects the article DOI: 10.1371/journal.pbio.1002564.].

Associative Mechanisms Allow for Social Learning and Cultural Transmission of String Pulling in an Insect.

Social insects make elaborate use of simple mechanisms to achieve seemingly complex behavior and may thus provide a unique resource to discover the basic cognitive elements required for culture, i.e., group-specific behaviors that spread from "innovators" to others in the group via social learning. We first explored whether bumblebees can learn a nonnatural object manipulation task by using string pulling to access a reward that was presented out of reach. Only a small minority "innovated" and solved the task spontaneously, but most bees were able to learn to pull a string when trained in a stepwise manner. In addition, naïve bees learnt the task by observing a trained demonstrator from a distance. Learning the behavior relied on a combination of simple associative mechanisms and trial-and-error learning and did not require "insight": naïve bees failed a "coiled-string experiment," in which they did not receive instant visual feedback of the target moving closer when tugging on the string. In cultural diffusion experiments, the skill spread rapidly from a single knowledgeable individual to the majority of a colony's foragers. We observed that there were several sequential sets ("generations") of learners, so that previously naïve observers could first acquire the technique by interacting with skilled individuals and, subsequently, themselves become demonstrators for the next "generation" of learners, so that the longevity of the skill in the population could outlast the lives of informed foragers. This suggests that, so long as animals have a basic toolkit of associative and motor learning processes, the key ingredients for the cultural spread of unusual skills are already in place and do not require sophisticated cognition.

Rapid behavioral maturation accelerates failure of stressed honey bee colonies.

Many complex factors have been linked to the recent marked increase in honey bee colony failure, including pests and pathogens, agrochemicals, and nutritional stressors. It remains unclear, however, why colonies frequently react to stressors by losing almost their entire adult bee population in a short time, resulting in a colony population collapse. Here we examine the social dynamics underlying such dramatic colony failure. Bees respond to many stressors by foraging earlier in life. We manipulated the demography of experimental colonies to induce precocious foraging in bees and used radio tag tracking to examine the consequences of precocious foraging for their performance. Precocious foragers completed far fewer foraging trips in their life, and had a higher risk of death in their first flights. We constructed a demographic model to explore how this individual reaction of bees to stress might impact colony performance. In the model, when forager death rates were chronically elevated, an increasingly younger forager force caused a positive feedback that dramatically accelerated terminal population decline in the colony. This resulted in a breakdown in division of labor and loss of the adult population, leaving only brood, food, and few adults in the hive. This study explains the social processes that drive rapid depopulation of a colony, and we explore possible strategies to prevent colony failure. Understanding the process of colony failure helps identify the most effective strategies to improve colony resilience.

A possible structural correlate of learning performance on a colour discrimination task in the brain of the bumblebee.

Synaptic plasticity is considered to be a basis for learning and memory. However, the relationship between synaptic arrangements and individual differences in learning and memory is poorly understood. Here, we explored how the density of microglomeruli (synaptic complexes) within specific regions of the bumblebee (Bombus terrestris) brain relates to both visual learning and inter-individual differences in learning and memory performance on a visual discrimination task. Using whole-brain immunolabelling, we measured the density of microglomeruli in the collar region (visual association areas) of the mushroom bodies of the bumblebee brain. We found that bumblebees which made fewer errors during training in a visual discrimination task had higher microglomerular density. Similarly, bumblebees that had better retention of the learned colour-reward associations two days after training had higher microglomerular density. Further experiments indicated experience-dependent changes in neural circuitry: learning a colour-reward contingency with 10 colours (but not two colours) does result, and exposure to many different colours may result, in changes to microglomerular density in the collar region of the mushroom bodies. These results reveal the varying roles that visual experience, visual learning and foraging activity have on neural structure. Although our study does not provide a causal link between microglomerular density and performance, the observed positive correlations provide new insights for future studies into how neural structure may relate to inter-individual differences in learning and memory.

Studying emotion in invertebrates: what has been done, what can be measured and what they can provide.

Until recently, whether invertebrates might exhibit emotions was unknown. This possibility has traditionally been dismissed by many as emotions are frequently defined with reference to human subjective experience, and invertebrates are often not considered to have the neural requirements for such sophisticated abilities. However, emotions are understood in humans and other vertebrates to be multifaceted brain states, comprising dissociable subjective, cognitive, behavioural and physiological components. In addition, accumulating literature is providing evidence of the impressive cognitive capacities and behavioural flexibility of invertebrates. Alongside these, within the past few years, a number of studies have adapted methods for assessing emotions in humans and other animals, to invertebrates, with intriguing results. Sea slugs, bees, crayfish, snails, crabs, flies and ants have all been shown to display various cognitive, behavioural and/or physiological phenomena that indicate internal states reminiscent of what we consider to be emotions. Given the limited neural architecture of many invertebrates, and the powerful tools available within invertebrate research, these results provide new opportunities for unveiling the neural mechanisms behind emotions and open new avenues towards the pharmacological manipulation of emotion and its genetic dissection, with advantages for disease research and therapeutic drug discovery. Here, we review the increasing evidence that invertebrates display some form of emotion, discuss the various methods used for assessing emotions in invertebrates and consider what can be garnered from further emotion research on invertebrates in terms of the evolution and underlying neural basis of emotion in a comparative context.

Accelerated behavioural development changes fine-scale search behaviour and spatial memory in honey bees (Apis mellifera L.).

Normally, worker honey bees (Apis mellifera) begin foraging when more than 2 weeks old as adults, but if individual bees or the colony is stressed, bees often begin foraging precociously. Here, we examined whether bees that accelerated their behavioural development to begin foraging precociously differed from normal-aged foragers in cognitive performance. We used a social manipulation to generate precocious foragers from small experimental colonies and tested their performance in a free-flight visual reversal learning task, and a test of spatial memory. To assess spatial memory, bees were trained to learn the location of a small sucrose feeder within an array of three landmarks. In tests, the feeder and one landmark were removed and the search behaviour of the bees was recorded. Performance of precocious and normal-aged foragers did not differ in a visual reversal learning task, but the two groups showed a clear difference in spatial memory. Flight behaviour suggested normal-aged foragers were better able to infer the position of the removed landmark and feeder relative to the remaining landmarks than precocious foragers. Previous studies have documented the cognitive decline of old foragers, but this is the first suggestion of a cognitive deficit in young foragers. These data imply that worker honey bees continue their cognitive development during the adult stage. These findings may also help to explain why precocious foragers perform quite poorly as foragers and have a higher than normal loss rate.

Honey bees selectively avoid difficult choices.

Human decision-making strategies are strongly influenced by an awareness of certainty or uncertainty (a form of metacognition) to increase the chances of making a right choice. Humans seek more information and defer choosing when they realize they have insufficient information to make an accurate decision, but whether animals are aware of uncertainty is currently highly contentious. To explore this issue, we examined how honey bees (Apis mellifera) responded to a visual discrimination task that varied in difficulty between trials. Free-flying bees were rewarded for a correct choice, punished for an incorrect choice, or could avoid choosing by exiting the trial (opting out). Bees opted out more often on difficult trials, and opting out improved their proportion of successful trials. Bees could also transfer the concept of opting out to a novel task. Our data show that bees selectively avoid difficult tasks they lack the information to solve. This finding has been considered as evidence that nonhuman animals can assess the certainty of a predicted outcome, and bees' performance was comparable to that of primates in a similar paradigm. We discuss whether these behavioral results prove bees react to uncertainty or whether associative mechanisms can explain such findings. To better frame metacognition as an issue for neurobiological investigation, we propose a neurobiological hypothesis of uncertainty monitoring based on the known circuitry of the honey bee brain.

Negative impact of manganese on honeybee foraging.

Anthropogenic accumulation of metals such as manganese is a well-established health risk factor for vertebrates. By contrast, the long-term impact of these contaminants on invertebrates is mostly unknown. Here, we demonstrate that manganese ingestion alters brain biogenic amine levels in honeybees and fruit flies. Furthermore, we show that manganese exposure negatively affects foraging behaviour in the honeybee, an economically important pollinator. Our findings indicate that in addition to its direct impact on human health, the common industrial contaminant manganese might also have indirect environmental and economical impacts via the modulation of neuronal and behavioural functions in economically important insects.

Peak shift in honey bee olfactory learning.

If animals are trained with two similar stimuli such that one is rewarding (S+) and one punishing (S-), then following training animals show a greatest preference not for the S+, but for a novel stimulus that is slightly more different from the S- than the S+ is. This peak shift phenomenon has been widely reported for vertebrates and has recently been demonstrated for bumblebees and honey bees. To explore the nature of peak shift in invertebrates further, here we examined the properties of peak shift in honey bees trained in a free-flight olfactory learning assay. Hexanal and heptanol were mixed in different ratios to create a continuum of odour stimuli. Bees were trained to artificial flowers such that one odour mixture was rewarded with 2 molar sucrose (S+), and one punished with distasteful quinine (S-). After training, bees were given a non-rewarded preference test with five different mixtures of hexanal and heptanol. Following training bees' maximal preference was for an odour mixture slightly more distinct from the S- than the trained S+. This effect was not seen if bees were initially trained with two distinct odours, replicating the classic features of peak shift reported for vertebrates. We propose a conceptual model of how peak shift might occur in honey bees. We argue that peak shift does not require any higher level of processing than the known olfactory learning circuitry of the bee brain and suggest that peak shift is a very general feature of discrimination learning.

Neural mechanisms of reward in insects.

Reward seeking is a major motivator and organizer of behavior, and animals readily learn to modify their behavior to more easily obtain reward, or to respond to stimuli that are predictive of reward. Here, we compare what is known of reward processing mechanisms in insects with the well-studied vertebrate reward systems. In insects almost all of what is known of reward processing is derived from studies of reward learning. This is localized to the mushroom bodies and antennal lobes and organized by a network of hierarchically arranged modulatory circuits, especially those involving octopamine and dopamine. Neurogenetic studies with Drosophila have identified distinct circuit elements for reward learning, "wanting," and possibly "liking" in Drosophila, suggesting a modular structure to the insect reward processing system, which broadly parallels that of the mammals in terms of functional organization.

Rats can make relative perceptual judgments about sequential stimuli.

In their natural environment, animals often make decisions based on abstract relationships among multiple stimulus representations. Humans and other primates can determine not only whether a sensory stimulus differs from a remembered sensory representation, but also how they differ along a particular dimension. However, much remains unknown about how such relative comparisons are made, and which species share this capacity, in part because most studies of sensory-guided decision making have utilized instrumental tasks in which choices are based on very simple stimulus-response associations. Here, we used a two-stimulus-interval discrimination task to test whether rats could determine how two sequentially presented stimuli were related along the dimension of odor quality (i.e., what the stimulus smells like). At a central port, rats sampled and compared two odor mixtures that consisted of spearmint and caraway in different ratios, separated by a 2-4-s interval, and then entered the left or right reward port. Water was delivered at the left if the first mixture consisted of more spearmint than the second did, and at the right otherwise. We found that the difference in mixture ratio predicted choice accuracy. Control experiments suggest that rats were indeed basing their choices on a comparison of odor quality across mixtures and were not using associative strategies. This study is the first demonstration of the use of a sequential "more than versus less than" rule in rats and provides a well-controlled paradigm for studying abstract comparisons in a rodent model system.

PKC{alpha}{beta}{gamma}- and PKC{delta}-dependent endocytosis of NBCe1-A and NBCe1-B in salivary parotid acinar cells.

We examined membrane trafficking of NBCe1-A and NBCe1-B variants of the electrogenic Na(+)-HCO(3)(-) cotransporter (NBCe1) encoded by the SLC4A4 gene, using confocal fluorescent microscopy in rat parotid acinar cells (ParC5 and ParC10). We showed that yellow fluorescent protein (YFP)-tagged NBCe1-A and green fluorescent protein (GFP)-tagged NBCe1-B are colocalized with E-cadherin in the basolateral membrane (BLM) but not with the apical membrane marker zona occludens 1 (ZO-1). We inhibited constitutive recycling with monensin and W13 and detected that NBCe1-A and NBCe1-B accumulated in vesicles marked with the early endosomal marker early endosome antigen-1 (EEA1), with a parallel loss from the BLM. We observed that NBCe1-A and NBCe1-B undergo massive carbachol (CCh)-stimulated redistribution from the BLM into early endosomes. We showed that internalization of NBCe1-A and NBCe1-B was prevented by the general PKC inhibitor GF-109203X, the PKCalphabetagamma-specific inhibitor Gö-6976, and the PKCdelta-specific inhibitor rottlerin. We verified the involvement of PKCdelta by blocking CCh-induced internalization of NBCe1-A-cyan fluorescent protein (CFP) in cells transfected with dominant-negative kinase-dead (Lys376Arg) PKCdelta-GFP. Our data suggest that NBCe1-A and NBCe1-B undergo constitutive and CCh-stimulated endocytosis regulated by conventional PKCs (PKCalphabetagamma) and by novel PKCdelta in rat epithelial cells. To help develop a more complete model of the role of NBCe1 in parotid acinar cells we also investigated the initial phase of the secretory response to cholinergic agonist. In an Ussing chamber study we showed that inhibition of basolateral NBCe1 with 5-chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-1H-indole-1-carboxamide (tenidap) significantly decreases an initial phase of luminal anion secretion measured as a transient short-circuit current (I(sc)) across ParC10 cell monolayers. Using trafficking and functional data we propose a model that describes a physiological role of NBC in salivary acinar cell secretion.

How foresight might support the behavioral flexibility of arthropods.

The small brains of insects and other invertebrates are often thought to constrain these animals to live entirely 'in the moment'. In this view, each one of their many seemingly hard-wired behavioral routines is triggered by a precisely defined environmental stimulus configuration, but there is no mental appreciation of the possible outcomes of one's actions, and therefore little flexibility. However, many studies show problem-solving behavior in various arthropod species that falls outside the range of fixed behavior routines. We propose that a basic form of foresight, the ability to predict the outcomes of one's own actions, is at the heart of such behavioral flexibility, and that the evolutionary roots of such outcome expectation are found in the need to disentangle sensory input that is predictable from self-generated motion versus input generated by changes in the outside world. Based on this, locusts, grasshoppers, dragonflies and flies seem to use internal models of the surrounding world to tailor their actions adaptively to predict the imminent future. Honeybees and orb-weaving spiders appear to act towards a desired outcome of their respective constructions, and the genetically pre-programmed routines that govern these constructions are subordinate to achieving the desired goal. Jumping spiders seem to preplan their route to prey suggesting they recognize the spatial challenge and actions necessary to obtain prey. Bumblebees and ants utilize objects not encountered in the wild as types of tools to solve problems in a manner that suggests an awareness of the desired outcome. Here we speculate that it may be simpler, in terms of the required evolutionary changes, computation and neural architecture, for arthropods to recognize their goal and predict the outcomes of their actions towards that goal, rather than having a large number of pre-programmed behaviors necessary to account for their observed behavioral flexibility.

Temporal correlation of elevated PRMT1 gene expression with mushroom body neurogenesis during bumblebee brain development.

Neural development depends on the controlled proliferation and differentiation of neural precursors. In holometabolous insects, these processes must be coordinated during larval and pupal development. Recently, protein arginine methylation has come into focus as an important mechanism of controlling neural stem cell proliferation and differentiation in mammals. Whether a similar mechanism is at work in insects is unknown. We investigated this possibility by determining the expression pattern of three protein arginine methyltransferase mRNAs (PRMT1, 4 and 5) in the developing brain of bumblebees by in situ hybridisation. We detected expression in neural precursors and neurons in functionally important brain areas throughout development. We found markedly higher expression of PRMT1, but not PRMT4 and PRMT5, in regions of mushroom bodies containing dividing cells during pupal stages at the time of active neurogenesis within this brain area. At later stages of development, PRMT1 expression levels were found to be uniform and did not correlate with actively dividing cells. Our study suggests a role for PRMT1 in regulating neural precursor divisions in the mushroom bodies of bumblebees during the period of neurogenesis.

Harmonic radar tracking reveals random dispersal pattern of bumblebee (Bombus terrestris) queens after hibernation.

The dispersal of animals from their birth place has profound effects on the immediate survival and longer-term persistence of populations. Molecular studies have estimated that bumblebee colonies can be established many kilometers from their queens' natal nest site. However, little is known about when and how queens disperse during their lifespan. One possible life stage when dispersal may occur, is directly after emerging from hibernation. Here, harmonic radar tracking of artificially over-wintered Bombus terrestris queens shows that they spend most of their time resting on the ground with intermittent very short flights (duration and distance). We corroborate these behaviors with observations of wild queen bees, which show similar prolonged resting periods between short flights, indicating that the behavior of our radar-monitored bees was not due to the attachment of transponders nor an artifact of the bees being commercially reared. Radar-monitored flights were not continuously directed away from the origin, suggesting that bees were not intentionally trying to disperse from their artificial emergence site. Flights did not loop back to the origin suggesting bees were not trying to remember or get back to the original release site. Most individuals dispersed from the range of the harmonic radar within less than two days and did not return. Flight directions were not different from a uniform distribution and flight lengths followed an exponential distribution, both suggesting random dispersal. A random walk model based on our observed data estimates a positive net dispersal from the origin over many flights, indicating a biased random dispersal, and estimates the net displacement of queens to be within the range of those estimated in genetic studies. We suggest that a distinct post-hibernation life history stage consisting mostly of rest with intermittent short flights and infrequent foraging fulfils the dual purpose of ovary development and dispersal prior to nest searching.

Honey bees increase their foraging performance and frequency of pollen trips through experience.

Honey bee foragers must supply their colony with a balance of pollen and nectar to sustain optimal colony development. Inter-individual behavioural variability among foragers is observed in terms of activity levels and nectar vs. pollen collection, however the causes of such variation are still open questions. Here we explored the relationship between foraging activity and foraging performance in honey bees (Apis mellifera) by using an automated behaviour monitoring system to record mass on departing the hive, trip duration, presence of pollen on the hind legs and mass upon return to the hive, during the lifelong foraging career of individual bees. In our colonies, only a subset of foragers collected pollen, and no bee exclusively foraged for pollen. A minority of very active bees (19% of the foragers) performed 50% of the colony's total foraging trips, contributing to both pollen and nectar collection. Foraging performance (amount and rate of food collection) depended on bees' individual experience (amount of foraging trips completed). We argue that this reveals an important vulnerability for these social bees since environmental stressors that alter the activity and reduce the lifespan of foragers may prevent bees ever achieving maximal performance, thereby seriously compromising the effectiveness of the colony foraging force.

Large-scale transcriptome changes in the process of long-term visual memory formation in the bumblebee, Bombus terrestris.

Many genes have been implicated in mechanisms of long-term memory formation, but there is still much to be learnt about how the genome dynamically responds, transcriptionally, during memory formation. In this study, we used high-throughput sequencing to examine how transcriptome profiles change during visual memory formation in the bumblebee (Bombus terrestris). Expression of fifty-five genes changed immediately after bees were trained to associate reward with a single coloured chip, and the upregulated genes were predominantly genes known to be involved in signal transduction. Changes in the expression of eighty-one genes were observed four hours after learning a new colour, and the majority of these were upregulated and related to transcription and translation, which suggests that the building of new proteins may be the predominant activity four hours after training. Several of the genes identified in this study (e.g. Rab10, Shank1 and Arhgap44) are interesting candidates for further investigation of the molecular mechanisms of long-term memory formation. Our data demonstrate the dynamic gene expression changes after associative colour learning and identify genes involved in each transcriptional wave, which will be useful for future studies of gene regulation in learning and long-term memory formation.

Invertebrate learning and cognition: relating phenomena to neural substrate.

Diverse invertebrate species have been used for studies of learning and comparative cognition. Although we have gained invaluable information from this, in this study we argue that our approach to comparative learning research is rather deficient. Generally invertebrate learning research has focused mainly on arthropods, and most of that within the Hymenoptera and Diptera. Any true comparative analysis of the distribution of comparative cognitive abilities across phyla is hampered by this bias, and more fundamentally by a reporting bias toward positive results. To understand the limits of learning and cognition for a species, knowing what animals cannot do is at least as important as reporting what they can. Finally, much more effort needs to be focused on the neurobiological analysis of different types of learning to truly understand the differences and similarities of learning types. In this review, we first give a brief overview of the various forms of learning in invertebrates. We also suggest areas where further study is needed for a more comparative understanding of learning. Finally, using what is known of learning in honeybees and the well-studied honeybee brain, we present a model of how various complex forms of learning may be accounted for with the same neural circuitry required for so-called simple learning types. At the neurobiological level, different learning phenomena are unlikely to be independent, and without considering this it is very difficult to correctly interpret the phylogenetic distribution of learning and cognitive abilities. WIREs Cogn Sci 2013, 4:561-582. doi: 10.1002/wcs.1248 For further resources related to this article, please visit the WIREs website.

Electrogenic NBCe1 (SLC4A4), but not electroneutral NBCn1 (SLC4A7), cotransporter undergoes cholinergic-stimulated endocytosis in salivary ParC5 cells.

Cholinergic agonists are major stimuli for fluid secretion in parotid acinar cells. Saliva bicarbonate is essential for maintaining oral health. Electrogenic and electroneutral Na(+)-HCO(3)(-) cotransporters (NBCe1 and NBCn1) are abundant in parotid glands. We previously reported that angiotensin regulates NBCe1 by endocytosis in Xenopus oocytes. Here, we studied cholinergic regulation of NBCe1 and NBCn1 membrane trafficking by confocal fluorescent microscopy and surface biotinylation in parotid epithelial cells. NBCe1 and NBCn1 colocalized with E-cadherin monoclonal antibody at the basolateral membrane (BLM) in polarized ParC5 cells. Inhibition of constitutive recycling with the carboxylic ionophore monensin or the calmodulin antagonist W-13 caused NBCe1 to accumulate in early endosomes with a parallel loss from the BLM, suggesting that NBCe1 is constitutively endocytosed. Carbachol and PMA likewise caused redistribution of NBCe1 from BLM to early endosomes. The PKC inhibitor, GF-109203X, blocked this redistribution, indicating a role for PKC. In contrast, BLM NBCn1 was not downregulated in parotid acinar cells treated with constitutive recycling inhibitors, cholinergic stimulators, or PMA. We likewise demonstrate striking differences in regulation of membrane trafficking of NBCe1 vs. NBCn1 in resting and stimulated cells. We speculate that endocytosis of NBCe1, which coincides with the transition to a steady-state phase of stimulated fluid secretion, could be a part of acinar cell adjustment to a continuous secretory response. Stable association of NBCn1 at the membrane may facilitate constitutive uptake of HCO(3)(-) across the BLM, thus supporting HCO(3)(-) luminal secretion and/or maintaining acid-base homeostasis in stimulated cells.