Periodic Reporting for period 1 - FEAR-BEES (Unravelling the neuro-ethological components of emotions in honey bees)
Reporting period: 2022-10-03 to 2024-10-02
The question whether invertebrates may have basic forms of emotions is relevant in the framework of current debates on the existence of sentience in these animals, i.e. the capacity of experiencing subjective feelings. A considerable body of research has already documented in a convincing way that several invertebrate species are capable of higher cognitive processing beyond simple associative learning. Yet, cognition does not mean emotion, and the fact that invertebrates are considered now from a more cognitive perspective than before, has in principle no implication for the presence of emotional processes. It is, however, undeniable that the change in perspective concerning the cognitive sophistication of invertebrates has enhanced awareness about the possibility that these animals may experience “emotional states”. An example in favour of this possibility is provided by our work showing that bumblebees behavior is consistent with positive emotional states (optimistic-like) following consumption of an unexpected reward. However, the comprehension of the neuro-physiological underpinnings of these “emotional states” is still limited despite using the term “emotional states” when referring to changes in behavior, based on common physiological mechanisms identified in other species. This void constitutes a missed opportunity to overcome the discussions on the appropriateness of semantic labels applied to invertebrate behavior by determining to what extent neural architectures and neurotransmitters involved in invertebrate “emotions” coincide with those found in mammals, including humans. This project will adopt a multicomponent approach, including behavioural, neurophysiological and cognitive analyses to characterize potential emotional states in invertebrates. To achieve this goal, we will focus on “fear” in honey bees (Apis mellifera) given the tractability of these insects for all these levels of analysis. The current project will advance our knowledge on the presence of “emotional states” in honey bees, decrypting underlying neural mechanisms and the phylogenetic link between these mechanisms and those of mammals, including humans. We will focus on the emotion of “fear” because this it has been widely characterized in vertebrates. In the case of honey bees, to avoid subjective labelling of behavioural states, we will use specific aversive stimuli and contexts that trigger alarm responses. The underlying question is whether bees react to these stimuli in an “reflexive” way or whether there is a subjective component accompanying these reactions. The proposed project has a societal, economical, and scientific impact. From a societal perspective, determining whether or not invertebrates possess “emotions” will affect how we interact with these and other animals and will generate fruitful ethical discussions as to how to conduct animal research. The economic impact is related to the importance of bees as pollinators. More than 150 $ billion worth of annual global food production relies on pollinators and this production is currently endangered by agricultural practices, such as intense farming, long distance transportation, the use of pesticides. To what extent these practices induce also “negative emotions” in pollinators is a relevant question as such states could affect productivity and thus agricultural crops. From a fundamental scientific perspective, our findings will shed light on the phylogenetic basis of our own emotions, how and why they evolved and how the brain generates them. The neurobiological study of emotions in animals can also have important consequences for improving treatment of affective disorders.
We had three major objectives with the overall aim of advancing our understanding of emotional states in honey bees, focusing on the neural mechanisms underlying these states and their phylogenetic link to those of mammals, including humans. We specifically selected the emotion of fear for this study, as fear responses are well-characterized in vertebrates. Our central question was whether bees react to fear-inducing stimuli in a purely "reflexive" manner or whether there is a subjective component accompanying these reactions. To address this, we outlined three work packages. In Objective 1, we characterized the behavioural and physiological correlates of fear in honey bees. The bees were subjected to a nociceptive stimulus (electric shock) within a classical conditioning protocol that allowed them to form expectations about that stimulus. We developed and refined a protocol (originally used at the host institution, ICARUS) that pairs a colour signal with an electric shock in a two-chamber compartment. Our results showed that bees not only learned the association between a light and the negative event but also retained this negative association for up to one hour. Additionally, we found that the intensity of the shock (5, 20, and 60 µA) modulated their behaviour (slowing or speeding the learning rate of individuals). This behavioural characterization was made possible through a custom-made Python script that allowed us to track and subsequently extract fine movements of the bees under testing conditions. Furthermore, we discovered that bees that learned the association exhibited higher CO2 emissions and thorax temperatures compared to control groups, i.e. bees that did not receive a shock when approaching the light or bees that received a shock that was not temporally contingent with the light. Overall, across six experiments that combined behavioural and physiological measures, the observed patterns strongly suggest that the bees' reactions were not simply reflexive. Subsequently, in Objective 2, we aimed to identify the molecular cascades of fear. We performed brain dissection on the samples collected during Objective 1 by dividing the brain in two main regions: the central brain and the optical lobes. Using High-Performance Liquid Chromatography (HPLC) we quantified the amount of Dopamine (DA), Serotonin (5HT) and Octopamine (OA) in the brain of 60 honey bees. We found that bees that learned the association between the light and the negative event have elevated amount of Serotonin and Octopamine compared to the control groups, indicating the potential roles of these neurotransmitters in the emotional processing. We further explored the role OA and 5HT in the emotional process of fear by conducting extensive pharmacological experiments. Depending on the experiment, we topically treated the bees’ brain with a specific drug, before testing (as described in Objective 1). Based on the HPLC analysis, we decided to focus on three main drugs, known to specifically block the octopaminergic and serotonergic systems. In detail, we conducted an initial experiment in which bees were topically treated with a drop of 1 µL of drugs on the cuticle, 30 minutes before conditioning. Bees were divided into nine groups: a No Shock group, Unpaired and Paired groups treated only with the solvent (Dimethylformamide) as controls, Paired groups treated with Epinastine (OA antagonist) at two different concentrations (1 and 10 nM), and Paired groups treated with a cocktail of Ketanserin and Methiotepin (5-HT antagonists) at two different concentrations (1 and 10 nM); finally, a Paired group treated only with Methiotepin at two different concentrations (1 and 10 nM). We found that bees treated with the Serotonin antagonist failed to learn the task. The involvement of Serotonin, a neurotransmitter widely known to be involved in mammalian emotions, highlights the phylogenetic link between invertebrate and vertebrate neurobiology.
Overall, we have characterised the basic emotion of fear in an invertebrate species, specifically honey bees, using a multidimensional approach. This was accomplished by integrating, on one side, well-developed methods applied in mammals, especially to farm animals, where different behavioural, cognitive, and neurophysiological assessments are combined to make indirect inferences about internal states that humans semantically identify as fear. On the other side, we selected honey bees as a model. This is a strategically important choice because bees exhibit sophisticated cognitive abilities, can be easily trained in the laboratory under a variety of conditioning protocols addressing different sensory modalities, and possess a nervous system accessible to several invasive techniques. This unique combination of advantages has been crucial for the progress of the project and to unravel the neuroethological basis of fear.
During the project we generated several innovative scientific outputs. We have developed and refined the ICARUS experimental apparatus, which was available at the host institution. We have developed an automatic tracking system synchronised to the ICARUS to allow tracking and extrapolation of fine behavioural measurements. We have also implemented a sound activity monitoring system, during testing.
The project has potential societal and economic impact. This impact is tied with the role that pollinators play in global biodiversity and food production. Pollinators contribute to over $150 billion worth of global food production, yet current agricultural pest management practices, such as pesticide use, pose significant risks to their health. Investigating whether these practices can induce negative emotional states in pollinators, like honey bees, could have direct implications for both their well-being and agricultural productivity. On a broader societal level, the recognition of emotional states in invertebrates and other non-human animals influences how we interact with them nudging debates around human-animal interactions across scientists, policy makers, stockholders and the general public.
The project is likely to impact my progression as an independent researcher. I have applied for a substantial grant (€300,000) provided by the Italian Ministry of University and Research (MUR) to researchers who were awarded a Marie Skłodowska-Curie Postdoctoral Fellowship. The purpose of the grant is to extend and/or develop a new research program based on the Marie Skłodowska-Curie project. In the new proposed project, I will investigate the social aspect of emotion investigating the emotional contagion of fear in honey bees. If successful, I will be working at the University of Padova in the Department of General Psychology for three years, starting in 2025.
During the project we generated several innovative scientific outputs. We have developed and refined the ICARUS experimental apparatus, which was available at the host institution. We have developed an automatic tracking system synchronised to the ICARUS to allow tracking and extrapolation of fine behavioural measurements. We have also implemented a sound activity monitoring system, during testing.
The project has potential societal and economic impact. This impact is tied with the role that pollinators play in global biodiversity and food production. Pollinators contribute to over $150 billion worth of global food production, yet current agricultural pest management practices, such as pesticide use, pose significant risks to their health. Investigating whether these practices can induce negative emotional states in pollinators, like honey bees, could have direct implications for both their well-being and agricultural productivity. On a broader societal level, the recognition of emotional states in invertebrates and other non-human animals influences how we interact with them nudging debates around human-animal interactions across scientists, policy makers, stockholders and the general public.
The project is likely to impact my progression as an independent researcher. I have applied for a substantial grant (€300,000) provided by the Italian Ministry of University and Research (MUR) to researchers who were awarded a Marie Skłodowska-Curie Postdoctoral Fellowship. The purpose of the grant is to extend and/or develop a new research program based on the Marie Skłodowska-Curie project. In the new proposed project, I will investigate the social aspect of emotion investigating the emotional contagion of fear in honey bees. If successful, I will be working at the University of Padova in the Department of General Psychology for three years, starting in 2025.