Andrés Arenas

Early olfactory experience modifies neural activity in the antennal lobe of a social insect at the adult stage

In the antennal lobe (AL), the first olfactory centre of the insect brain, odorants are represented as spatiotemporal patterns of glomerular activity. Whether and how such patterns are modified in the long term after precocious olfactory experiences (i.e. in the first days of adulthood) remains unknown. To address this question, we used in vivo optical imaging of calcium activity in the antennal lobe of 17‐day‐old honeybees which either experienced an odorant associated with sucrose solution 5–8 days after emergence or were left untreated. In both cases, we imaged neural responses to the learned odor and to three novel odors varying in functional group and carbon‐chain length. Two different odor concentrations were used. We also measured behavioral responses of 17‐day‐old honeybees, treated and untreated, to these stimuli. We show that precocious olfactory experience increased general odor‐induced activity and the number of activated glomeruli in the adult AL, but also affected qualitative odor representations, which appeared shifted in the neural space of treated animals relative to control animals. Such effects were not limited to the experienced odor, but were generalized to other perceptually similar odors. A similar trend was found in behavioral experiments, in which increased responses to the learned odor extended to perceptually similar odors in treated bees. Our results show that early olfactory experiences have long‐lasting effects, reflected in behavioral responses to odorants and concomitant neural activity in the adult olfactory system.

Early olfactory experience induces structural changes in the primary olfactory center of an insect brain

The antennal lobe (AL) is the first olfactory center of the insect brain and is constituted of different functional units, the glomeruli. In the AL, odors are coded as spatiotemporal patterns of glomerular activity. In honeybees, olfactory learning during early adulthood modifies neural activity in the AL on a long‐term scale and also enhances later memory retention. By means of behavioral experiments, we first verified that olfactory learning between the fifth and eighth day of adulthood induces better retention performances at a late adult stage than the same experience acquired before or after this period. We checked that the specificity of memory for the odorants used was improved. We then studied whether such early olfactory learning also induces long‐term structural changes in the AL consistent with the formation of long‐term olfactory memories. We also measured the volume of 15 identified glomeruli in the ALs of 17‐day‐old honeybees that either experienced an odor associated with sucrose solution between the fifth and eighth day of adulthood or were left untreated. We found that early olfactory experience induces glomerulus‐selective increases in volume that were specific to the learned odor. By comparing our volumetric measures with calcium‐imaging recordings from a previous study, performed in 17‐day‐old bees subjected to the same treatment and experimental conditions, we found that glomeruli that showed structural changes after early learning were those that exhibited a significant increase in neural activity. Our results make evident a correlation between structural and functional changes in the AL following early olfactory learning.

Floral scents experienced within the colony affect long-term foraging preferences in honeybees

Food scents circulating inside beehives influence foraging preferences in the field. However, the persistence and nature of the prior experience that lead to this biased response remain unknown. To determine how long honeybees show a food preference after experiencing a scented food inside the colony, we offered sugar solution scented into beehives. After all the combs of the experimental colonies were replaced, the landing responses of foragers were quantified in a two-scented-feeder situation outside the colony. We also tested whether bees exposed to a volatile compound inside the hive preferred a feeder scented with this odor to an unscented one. Results support the conclusion that the offering of a scented food enhanced the bias to this odor, whereas the volatile compound exposure caused a reduced landing response towards the scented option. These results suggest that olfactory experiences occurring within the beehive can promote changes in foraging preferences for several days.ZusammenfassungBei Honigbienen ist die Bewertung eines spezifischen Reizes als Anzeiger einer Belohnung stark von dem Kontext abhängig, in dem dieser wahrgenommen wurde (Sandoz et al., 2000; Gerber et al., 1996). Um das Sammelverhalten zu optimieren, lernen Honigbienen sowohl an der Sammelstelle als auch innerhalb des Volkes olfaktorische Blüteneigenschaften mit einer Belohnung zu assoziieren (z.B. von Frisch, 1923; Wenner et al., 1969). In neueren Untersuchungen konnten wir zeigen, dass assoziative Lernereignisse wie die obengenannten zur Ausbildung eines olfaktorischen Gedächtnisses führen, das dann wiederum dazu genutzt wird, den gelernten Duft in der Umgebung aufzufinden (Arenas et al., 2007). Hierbei zeigten Bienen mehrere Stunden nachdem sie mit duftender Nahrung behandelt worden waren eine Präferenz für diesen Duft. Dies war auch dann der Fall, wenn sie aus Völkern kamen, aus denen die Vorräte vollständig entfernt worden waren. Es war allerdings unbekannt, über welche Zeit hinweg diese Präferenz beibehalten wird. Daher boten wir den Bienen über vier Tage hinweg eine mit einem reinen Duft (Linalool oder Phenylacetaldehyde) versehene Zuckerlösung innerhalb des Bienenvolkes an. Danach wurden die Landeereignisse der Sammlerinnen in einer mit zwei verschiedenen Gerüchen markierten Fütterungseinrichtung außerhalb des Volkes registriert. Die Sammlerinnen zeigten eine Bevorzugung des zuvor in Umlauf gebrachten Futterduftes über vier Tage nach Entfernung aller Futtervorräte aus dem Bienenvolk (Abb. 1). Einer der Düfte (Linalool) beeinflusste die Landeereignisse über bis zu 8 Tage nach Entfernung aller Futtervorräte (Abb. 1).Da das Anbieten bedufteten Futters in sich selbst ein Sinnesereignis darstellt, das das Sammelverhalten beeinflussen könnte (z.B. durch sensorisches “priming”), führten wir in zweites Experiment durch. In diesem wollten wir herausfinden, ob die Futterpräferenzen auch dann bestanden wenn der Duft in der Lösung nur verdünnt vorhanden war oder innerhalb des Volkes angeboten wurde, aber nicht in der Futterlösung. Dann untersuchten wir zu verschiedenen Zeiten (vor, während und nach der Geruchserfahrung), ob die Sammlerinnen den bereits wahrgenommenen Geruch gegenüber einer geruchslosen Wahlalternative bevorzugten. Wir beobachteten eine Verringerung in der Anzahl der Bienen, die auf dem Fütterer mit bekanntem Geruch landeten, während die Landungen auf dem Fütterer mit einem neuen Geruch gleich blieben. Hierdurch zeigten wir, dass die Beeinflussung der Futterwahl bei Honigbienen von mehreren Faktoren abhängt: (i) Die geruchlichen Erfahrungen innerhalb des Volkes bauen Gedächtnisinhalte auf, die über lange Zeit bestehen bleiben, auch wenn die Bienen Sammelzugang zu natürlichen Futterquellen haben. (ii) Die unbelohnten Geruchserfahrungen innerhalb des Bienenvolkes beeinflussen die Futterwahl außerhalb des Bienenvolkes im entgegengesetzten Sinne wie bei i). (iii) Je nach angewendeter Methodik können daher unterschiedliche Ergebnisse resultieren.

Learned olfactory cues affect pollen-foraging preferences in honeybees, Apis mellifera

Honeybees, Apis mellifera, show learned odour preferences for flowers that provide nectar as a reward. However, little is known about such behavioural plasticity when bees exploit pollen sources. Furthermore, the question about whether nectar and pollen foragers use the same learned strategy to improve foraging efficiency remains untested. Here, we demonstrate that honeybee foragers are able to learn odour cues associated with pollen as a reward. This was tested in free-flying bees in a dual-choice feeding device after the bees had gathered pollen from a scented feeder. Free-flying bees that associated odour with pollen successfully recalled these memories in olfactometer odour choice tests in a Y-maze, but they failed to show extension of the proboscis to learned odour cues when restrained (proboscis extension reflex, PER, assay). In addition, odour cues associated with pollen at the feeding site induced foraging reactivation when bees were blown into the hive. In PER assays, after fatty acids were applied to the bees’ antennae, pollen foragers were more responsive than nectar foragers. This, in turn, allowed pollen foragers in the PER assay to associate an odour cue with pollen in some trials. On the other hand, the unconditioned response (UR) and the odour-conditioned response (CR) to sucrose and amino acids were similar for both types of foragers. Pollen foragers also showed more URs to fresh pollen of different flower species and even performed better during conditioning with some pollen types as the reward than did nectar foragers. By studying biases in pollen-foraging responses after learning, we provide new insights to help comprehend and characterize the search for food between pollen and nonpollen honeybee foragers.

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.

Learning through the waste: olfactory cues from the colony refuse influence plant preferences in foraging leaf-cutting ants

ABSTRACT Leaf-cutting ants learn to avoid plants initially harvested if they prove to be harmful for their symbiotic fungus once incorporated into the nest. At this point, waste particles removed from the fungus garden are likely to contain cues originating from both the unsuitable plant and the damaged fungus. We investigated whether leaf-cutting ant foragers learn to avoid unsuitable plants solely through the colony waste. We fed subcolonies of Acromymex ambiguus privet leaves treated with a fungicide undetectable to the ants, then collected the produced waste, and placed it into the fungus chamber of naive subcolonies. In individual choice tests, naive foragers preferred privet leaves before waste was put into the fungus chamber, but avoided them afterwards. Evidence on the influence of olfactory cues from the waste on decision making by foragers was obtained by scenting and transferring waste particles from subcolonies that had been fed either fungicide-treated or untreated leaves. In choice experiments, foragers from subcolonies given scented waste originating from fungicide-treated leaves collected fewer sugared paper discs with that scent compared with foragers from subcolonies given scented waste from untreated leaves. The results indicate that foragers learn to avoid plants unsuitable for the fungus by associating plant odours and cues from the damaged fungus that are present in waste particles. It is argued that waste particles may contribute to spread information about noxious plants for the fungus within the colony. Summary: Olfactory cues from the colony refuse influence plant preferences in foraging leaf-cutting ants.

Avoidance of plants unsuitable for the symbiotic fungus in leaf-cutting ants: Learning can take place entirely at the colony dump.

Plants initially accepted by foraging leaf-cutting ants are later avoided if they prove unsuitable for their symbiotic fungus. Plant avoidance is mediated by the waste produced in the fungus garden soon after the incorporation of the unsuitable leaves, as foragers can learn plant odors and cues from the damaged fungus that are both present in the recently produced waste particles. We asked whether avoidance learning of plants unsuitable for the symbiotic fungus can take place entirely at the colony dump. In order to investigate whether cues available in the waste chamber induce plant avoidance in naïve subcolonies, we exchanged the waste produced by subcolonies fed either fungicide-treated privet leaves or untreated leaves and measured the acceptance of untreated privet leaves before and after the exchange of waste. Second, we evaluated whether foragers could perceive the avoidance cues directly at the dump by quantifying the visits of labeled foragers to the waste chamber. Finally, we asked whether foragers learn to specifically avoid untreated leaves of a plant after a confinement over 3 hours in the dump of subcolonies that were previously fed fungicide-treated leaves of that species. After the exchange of the waste chambers, workers from subcolonies that had access to waste from fungicide-treated privet leaves learned to avoid that plant. One-third of the labeled foragers visited the dump. Furthermore, naïve foragers learned to avoid a specific, previously unsuitable plant if exposed solely to cues of the dump during confinement. We suggest that cues at the dump enable foragers to predict the unsuitable effects of plants even if they had never been experienced in the fungus garden.

Odor Experiences during Preimaginal Stages Cause Behavioral and Neural Plasticity in Adult Honeybees.

In eusocial insects, experiences acquired during the development have long-term consequences on mature behavior. In the honeybee that suffers profound changes associated with metamorphosis, the effect of odor experiences at larval instars on the subsequent physiological and behavioral response is still unclear. To address the impact of preimaginal experiences on the adult honeybee, colonies containing larvae were fed scented food. The effect of the preimaginal experiences with the food odor was assessed in learning performance, memory retention and generalization in 3–5- and 17–19 day-old bees, in the regulation of their expression of synaptic-related genes and in the perception and morphology of their antennae. Three-five day old bees that experienced 1-hexanol (1-HEX) as food scent responded more to the presentation of the odor during the 1-HEX conditioning than control bees (i.e., bees reared in colonies fed unscented food). Higher levels of proboscis extension response (PER) to 1-HEX in this group also extended to HEXA, the most perceptually similar odor to the experienced one that we tested. These results were not observed for the group tested at older ages. In the brain of young adults, larval experiences triggered similar levels of neurexins (NRXs) and neuroligins (Nlgs) expression, two proteins that have been involved in synaptic formation after associative learning. At the sensory periphery, the experience did not alter the number of the olfactory sensilla placoidea, but did reduce the electrical response of the antennae to the experienced and novel odor. Our study provides a new insight into the effects of preimaginal experiences in the honeybee and the mechanisms underlying olfactory plasticity at larval stage of holometabolous insects.