Effect of juvenile hormone on short-term olfactory memory in young honeybees (Apis mellifera).

Reliable retention of olfactory learning following a 1-trial classical conditioning of the proboscis extension reflex (PER) is not achieved in honeybees until they are 6-7 days old. Here we show that treatment of newly emerged honeybees with juvenile hormone (JH) has a profound effect on the maturation of short-term olfactory memory. JH-treated individuals display excellent short-term (1 h) memory of associative learning at times as early as 3 days of age and perform consistently better than untreated bees for at least the first week of their lives. By contrast, the retention of long-term (24 h) memory following a 3-trial conditioning of the PER is not significantly improved in JH-treated bees. Our study also shows that experience and (or) chemosensory activation are not essential to improve learning performance in olfactory tasks. The lack of accelerated development of long-term retention of olfactory memories in JH-treated honeybees is discussed in the context of neural circuits suspected to mediate memory formation and retrieval in the honeybee brain.     

Associative Learning during Early Adulthood Enhances Later Memory Retention in Honeybees

Background

Cognitive experiences during the early stages of life play an important role in shaping the future behavior in mammals but also in insects, in which precocious learning can directly modify behaviors later in life depending on both the timing and the rearing environment. However, whether olfactory associative learning acquired early in the adult stage of insects affect memorizing of new learning events has not been studied yet.

Methodology

Groups of adult honeybee workers that experienced an odor paired with a sucrose solution 5 to 8 days or 9 to 12 days after emergence were previously exposed to (i) a rewarded experience through the offering of scented food, or (ii) a non-rewarded experience with a pure volatile compound in the rearing environment.

Principal Findings

Early rewarded experiences (either at 1–4 or 5–8 days of adult age) enhanced retention performance in 9–12-day-conditioned bees when they were tested at 17 days of age. The highest retention levels at this age, which could not be improved with prior rewarded experiences, were found for memories established at 5–8 days of adult age. Associative memories acquired at 9–12 days of age showed a weak effect on retention for some pure pre-exposed volatile compounds; whereas the sole exposure of an odor at any younger age did not promote long-term effects on learning performance.

Conclusions

The associative learning events that occurred a few days after adult emergence improved memorizing in middle-aged bees. In addition, both the timing and the nature of early sensory inputs interact to enhance retention of new learning events acquired later in life, an important matter in the social life of honeybees.     

Olfactory information transfer in the honeybee: compared efficiency of classical conditioning and early exposure

We investigated the ability of honeybees, Apis mellifera, to use olfactory information gained in a given experimental context, in other contexts. First, restrained bees were subjected to a Pavlovian associative learning procedure, based on the conditioning of the proboscis extension response (PER), where a floral odour was paired with a sugar reward. We observed the orientation behaviour of conditioned and na?ve bees in a four-armed olfactometer with four contiguous fields either scented with the conditioning odour or unscented. Information transfer was clearly shown, conditioned bees orienting towards the conditioning odour, whilst na?ve bees shunned it. Second, the effect of passive olfactory exposures during the bees' development was assessed in two behavioural contexts: either orientation in the olfactometer or a PER conditioning procedure. Two exposure periods were applied: (1) the pupal stage (9 days before emergence); (2) the early adult stage (8 days after emergence). No effect of preimaginal exposure was recorded, but exposure during the early adult stage induced a higher choice frequency of the odour field in the olfactometer, and lower learning performance in the PER conditioning assay. These observations show that olfactory information gained during development can modify bees' later behaviour in different contexts: this is another instance of olfactory information transfer in bees. These results also suggest that nonassociative learning phenomena, taking place at a critical period during development, might be involved in the maturation of the bees' olfactory system, and in the organization of odour-mediated behaviours. Copyright 2000 The Association for the Study of Animal Behaviour.     

Neuronal Plasticity in the Mushroom‐Body Calyx of Bumble Bee Workers During Early Adult Development

Division of labor among workers is a key feature of social insects and frequently characterized by an age‐related transition between tasks, which is accompanied by considerable structural changes in higher brain centers. Bumble bees (Bombus terrestris), in contrast, exhibit a size‐related rather than an age‐related task allocation, and thus workers may already start foraging at two days of age. We ask how this early behavioral maturation and distinct size variation are represented at the neuronal level and focused our analysis on the mushroom bodies (MBs), brain centers associated with sensory integration, learning and memory. To test for structural neuronal changes related to age, light exposure, and body size, whole‐mount brains of age‐marked workers were dissected for synapsin immunolabeling. MB calyx volumes, densities, and absolute numbers of olfactory and visual projection neuron (PN) boutons were determined by confocal laser scanning microscopy and three‐dimensional image analyses. Dark‐reared bumble bee workers showed an early age‐related volume increase in olfactory and visual calyx subcompartments together with a decrease in PN‐bouton density during the first three days of adult life. A 12:12  h light‐dark cycle did not affect structural organization of the MB calyces compared to dark‐reared individuals. MB calyx volumes and bouton numbers positively correlated with body size, whereas bouton density was lower in larger workers. We conclude that, in comparison to the closely related honey bees, neuronal maturation in bumble bees is completed at a much earlier stage, suggesting a strong correlation between neuronal maturation time and lifestyle in both species.     

Olfactory interference during inhibitory backward pairing in honey bees

Background

Restrained worker honey bees are a valuable model for studying the behavioral and neural bases of olfactory plasticity. The proboscis extension response (PER; the proboscis is the mouthpart of honey bees) is released in response to sucrose stimulation. If sucrose stimulation is preceded one or a few times by an odor (forward pairing), the bee will form a memory for this association, and subsequent presentations of the odor alone are sufficient to elicit the PER. However, backward pairing between the two stimuli (sucrose, then odor) has not been studied to any great extent in bees, although the vertebrate literature indicates that it elicits a form of inhibitory plasticity.

Methodology/Principal Findings

If hungry bees are fed with sucrose, they will release a long lasting PER; however, this PER can be interrupted if an odor is presented 15 seconds (but not 7 or 30 seconds) after the sucrose (backward pairing). We refer to this previously unreported process as olfactory interference. Bees receiving this 15 second backward pairing show reduced performance after a subsequent single forward pairing (excitatory conditioning) trial. Analysis of the results supported a relationship between olfactory interference and a form of backward pairing-induced inhibitory learning/memory. Injecting the drug cimetidine into the deutocerebrum impaired olfactory interference.

Conclusions/Significance

Olfactory interference depends on the associative link between odor and PER, rather than between odor and sucrose. Furthermore, pairing an odor with sucrose can lead either to association of this odor to PER or to the inhibition of PER by this odor. Olfactory interference may provide insight into processes that gate how excitatory and inhibitory memories for odor-PER associations are formed.     

Odor information transfer in the stingless bee<Emphasis Type="Italic"> Melipona quadrifasciata</Emphasis>: effect of in-hive experiences on classical conditioning of proboscis extension

A recent study showed that the stingless bee Melipona quadrifasciata could learn to discriminate odors in a classical conditioning of proboscis extension response (PER). Here we used this protocol to investigate the ability of these bees to use olfactory information obtained within the colony in an experimental context: the PER paradigm. We compared their success in solving a classical differential conditioning depending on the previous olfactory experiences received inside the nest. We found that M. quadrifasciata bees are capable of transferring the food-odor information acquired in the colony to a differential conditioning in the PER paradigm. Bees attained higher discrimination levels when they had previously encountered the rewarded odor associated to food inside the hive. The increase in the discrimination levels, however, was in some cases unspecific to the odor used indicating a certain degree of generalization. The influence of the food scent offered at a field feeder 24 h before the classical conditioning could also be seen in the discrimination attained by the foragers in the PER setup, detecting the presence of long-term memory. Moreover, the improved performance of recruited bees in the PER paradigm suggests the occurrence of social learning of nectar scents inside the stingless bees’ hives.     

The influence of gustatory and olfactory experiences on responsiveness to reward in the honeybee

Background

Honeybees (Apis mellifera) exhibit an extraordinarily tuned division of labor that depends on age polyethism. This adjustment is generally associated with the fact that individuals of different ages display different response thresholds to given stimuli, which determine specific behaviors. For instance, the sucrose-response threshold (SRT) which largely depends on genetic factors may also be affected by the nectar sugar content. However, it remains unknown whether SRTs in workers of different ages and tasks can differ depending on gustatory and olfactory experiences.

Methodology

Groups of worker bees reared either in an artificial environment or else in a queen-right colony, were exposed to different reward conditions at different adult ages. Gustatory response scores (GRSs) and odor-memory retrieval were measured in bees that were previously exposed to changes in food characteristics.

Principal Findings

Results show that the gustatory responses of pre-foraging-aged bees are affected by changes in sucrose solution concentration and also to the presence of an odor provided it is presented as scented sucrose solution. In contrast no differences in worker responses were observed when presented with odor only in the rearing environment. Fast modulation of GRSs was observed in older bees (12–16 days of age) which are commonly involved in food processing tasks within the hive, while slower modulation times were observed in younger bees (commonly nurse bees, 6–9 days of age). This suggests that older food-processing bees have a higher plasticity when responding to fluctuations in resource information than younger hive bees. Adjustments in the number of trophallaxis events were also found when scented food circulated inside the nest, and this was positively correlated with the differences in timing observed in gustatory responsiveness and memory retention for hive bees of different age classes.

Conclusions

This work demonstrates the accessibility of chemosensory information in the honeybee colonies with respect to incoming nectar. The modulation of the sensory-response systems within the hive can have important effects on the dynamics of food transfer and information propagation.     

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee''s behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.     

Apis mellifera bees acquire long-term olfactory memories within the colony

Early studies indicate that Apis mellifera bees learn nectar odours within their colonies. This form of olfactory learning, however, has not been analysed by measuring well-quantifiable learning performances and the question remains whether it constitutes a 'robust' form of learning. Hence, we asked whether bees acquire long-term olfactory memories within the colony. To this end, we used the bee proboscis extension response. We found that within-the-nest bees do indeed associate the odour (as the conditioned stimulus) with the sugar (as the unconditioned stimulus) present in the incoming nectar, and that the distribution of scented nectar within the colony allows them to establish long-term olfactory memories. This finding is discussed in the context of efficient foraging.     

Learning impairment in honey bees caused by agricultural spray adjuvants

Background

Spray adjuvants are often applied to crops in conjunction with agricultural pesticides in order to boost the efficacy of the active ingredient(s). The adjuvants themselves are largely assumed to be biologically inert and are therefore subject to minimal scrutiny and toxicological testing by regulatory agencies. Honey bees are exposed to a wide array of pesticides as they conduct normal foraging operations, meaning that they are likely exposed to spray adjuvants as well. It was previously unknown whether these agrochemicals have any deleterious effects on honey bee behavior.

Methodology/Principal Findings

An improved, automated version of the proboscis extension reflex (PER) assay with a high degree of trial-to-trial reproducibility was used to measure the olfactory learning ability of honey bees treated orally with sublethal doses of the most widely used spray adjuvants on almonds in the Central Valley of California. Three different adjuvant classes (nonionic surfactants, crop oil concentrates, and organosilicone surfactants) were investigated in this study. Learning was impaired after ingestion of 20 µg organosilicone surfactant, indicating harmful effects on honey bees caused by agrochemicals previously believed to be innocuous. Organosilicones were more active than the nonionic adjuvants, while the crop oil concentrates were inactive. Ingestion was required for the tested adjuvant to have an effect on learning, as exposure via antennal contact only induced no level of impairment.

Conclusions/Significance

A decrease in percent conditioned response after ingestion of organosilicone surfactants has been demonstrated here for the first time. Olfactory learning is important for foraging honey bees because it allows them to exploit the most productive floral resources in an area at any given time. Impairment of this learning ability may have serious implications for foraging efficiency at the colony level, as well as potentially many social interactions. Organosilicone spray adjuvants may therefore contribute to the ongoing global decline in honey bee health.