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Ants have recently emerged as useful models for the study of olfactory learning. In this framework, the development of a protocol for the appetitive conditioning of the maxilla-labium extension response (MaLER) provided the possibility of studying Pavlovian odor-food learning in a controlled environment. Here we extend these studies by introducing the first Pavlovian aversive learning protocol for harnessed ants in the laboratory. We worked with carpenter ants Camponotus aethiops and first determined the capacity of different temperatures applied to the body surface to elicit the typical aversive mandible opening response (MOR). We determined that 75°C is the optimal temperature to induce MOR and chose the hind legs as the stimulated body region because of their high sensitivity. We then studied the ability of ants to learn and remember odor-heat associations using 75°C as the unconditioned stimulus. We studied learning and short-term retention after absolute (one odor paired with heat) and differential conditioning (a punished odor versus an unpunished odor). Our results show that ants successfully learn the odor-heat association under a differential-conditioning regime and thus exhibit a conditioned MOR to the punished odor. Yet, their performance under an absolute-conditioning regime is poor. These results demonstrate that ants are capable of aversive learning and confirm previous findings about the different attentional resources solicited by differential and absolute conditioning in general., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Memory is a dynamic process that allows encoding, storage, and retrieval of information acquired through individual experience. In the honeybee Apis mellifera, olfactory conditioning of the proboscis extension response (PER) has shown that besides short-term memory (STM) and mid-term memory (MTM), two phases of long-term memory (LTM) are formed upon multiple-trial conditioning: an early phase (e-LTM) which depends on translation from already available mRNA, and a late phase (l-LTM) which requires de novo transcription and translation. Here we combined olfactory PER conditioning and neuropharmacological inhibition and studied the involvement of the NO-cGMP pathway, and of specific molecules, such as cyclic nucleotide-gated channels (CNG), calmodulin (CaM), adenylyl cyclase (AC), and Ca(2+)/calmodulin-dependent protein kinase (CaMKII), in the formation of olfactory LTM in bees. We show that in addition to NO-cGMP and cAMP-PKA, CNG channels, CaM, AC, and CaMKII also participate in the formation of a l-LTM (72-h post-conditioning) that is specific for the learned odor. Importantly, the same molecules are dispensable for olfactory learning and for the formation of both MTM (in the minute and hour range) and e-LTM (24-h post-conditioning), thus suggesting that the signaling pathways leading to l-LTM or e-LTM involve different molecular actors.

Storage of information into long-term memory (LTM) usually requires at least two waves of transcription in many species. However, there is no clear evidence of this phenomenon in insects, which are influential models for memory studies. We measured retention in honeybees after injecting a transcription inhibitor at different times before and after conditioning. We identified two separate time windows during which the transcription blockade impairs memory quantitatively and qualitatively, suggesting the occurrence of an early transcription wave (triggered during conditioning) and a later one (starting several hours after learning). Hence insects, like other species, would require two transcription waves for LTM formation.

Latent inhibition (LI) is a decrement in learning performance that results from the nonreinforced pre-exposure of the to-be-conditioned stimulus, in both vertebrates and invertebrates. In vertebrates, LI development involves dopamine and serotonin; in invertebrates there is yet no information. We studied differential olfactory conditioning of the proboscis extension response in the honeybee Apis mellifera, and we compared LI in individuals treated with antagonists of biogenic amines (dopamine, octopamine, and serotonin). An antagonist of octopamine receptors and two antagonists of serotonin receptors showed LI disruption. We thus provide evidence that serotonin would participate in the regulation of LI in honeybees.

Olfactory learning and memory processes in Drosophila have been well investigated with aversive conditioning, but appetitive conditioning has rarely been documented. Here, we report for the first time individual olfactory conditioning of proboscis activity in restrained Drosophila melanogaster. The protocol was adapted from those developed for proboscis extension conditioning in the honeybee Apis mellifera. After establishing a scale of small proboscis movements necessary to characterize responses to olfactory stimulation, we applied Pavlovian conditioning, with five trials consisting of paired presentation of a banana odour and a sucrose reward. Drosophila showed conditioned proboscis activity to the odour, with a twofold increase of percentage of responses after the first trial. No change occurred in flies experiencing unpaired presentations of the stimuli, confirming an associative basis for this form of olfactory learning. The adenylyl cyclase mutant rutabaga did not exhibit learning in this paradigm. This protocol generated at least a short-term memory of 15 min, but no significant associative memory was detected at 1 h. We also showed that learning performance was dependent on food motivation, by comparing flies subjected to different starvation regimes.