DOI: 10.5061/dryad.hx3ffbgbc
title: Lateralisation of short- and long-term visual memories in an insect

description: The formation of memories within the vertebrate brain is lateralised
 between hemispheres across multiple modalities, however, in invertebrates
 evidence for lateralisation is restricted to olfactory memories, primarily
 from social bees. Here we use a classical conditioning paradigm with a
 visual conditioned stimulus to show that visual memories are lateralised
 in the wood ant, Formica rufa. We show that a brief contact between a
 sugar reward and either the right or left antenna (reinforcement) is
 sufficient to produce a lateralised memory, even though the visual cue is
 visible to both eyes throughout training and testing. Reinforcement given
 to the right antenna induced short-term memories whereas reinforcement
 given to the left antenna induced long-term memories. Thus, short- and
 long-term visual memories are lateralised in wood ants. This extends the
 modalities across which visual memories are lateralised in insects and
 suggests that such memory lateralisation may have evolved multiple times,
 possibly linked to the evolution of eusociality in the Hymenoptera.
description: (a) Animals and preparation<br> Red wood ant workers
 (Formica rufa L.) from three different colonies were used for the
 experiments. These colonies were collected from Ashdown Forest, Sussex, UK
 (N 51 4.680, E 0 1.800) in June 2017 and June and August 2018. They were
 maintained indoors for at least 2 months prior to the experiments
 commencing, during which they were kept at 26°C, under a 12 h light:12 h
 dark cycle and fed with sucrose (333 g/L). Prior to training, colonies
 were starved for at least 2 days to increase motivation for feeding. Ants
 were selected from the colony on the day of training and harnessed to a
 custom-made holder (see Fernandes et al., 2018). (b)
 Training<br> Ants were trained to associate a visual cue with a
 sucrose reward as described in Fernandes et al. (2018). We performed six
 different types of training to investigate the role of the antennae during
 visual associative learning. Paired Right (PR) and Paired Left (PL) ants
 experienced right or left antenna touching by the US prior to contact with
 the mouthparts, respectively. Paired Bilateral (PB) ants were trained with
 a sucrose reward to both antennae prior to feeding, and data pooled with
 that from Fernandes et al. (2018). No touch to the antennae (NTA) ants had
 their antenna attached to the insect pin with low melting temperature wax
 to prevent antennal contact with the reward (US) during
 training.<br> Unpaired controls were carried out as described in
 Fernandes et al. (2018). Unpaired Right (UPR) and Unpaired Left (UPL) ants
 experienced right or left antenna touching by the US prior to contact with
 the mouthparts, respectively. An additional unpaired group, Randomized
 Unpaired Left (RUPL), was trained identically to the UPL cohort except CS
 and US were presented in a randomized order. A CS-only (CSO) control group
 had the CS (plus syringe) presentation for the same number of trials, but
 without the US to antennae or mouthparts. A US-only (USO) control was
 given paired training but with the CS removed and replaced with a white
 circular cue (10 cm diameter) to remove all potential visual cues
 generated during CS delivery except the syringe tip and sucrose drop. The
 USO did not differ in memory formation from the CSO (supplementary table
 1) and is not further discussed here. For all types of training, half of
 the ants were trained with their right side facing the experimenter and
 the other half with the left, to account for potential environmental
 asymmetries that could be learnt during training. In both cases the visual
 stimulus faced the ant and was seen by both eyes.  (c)
 Testing<br> We tested all groups in the same set-up with a 10 second
 CS presentation either 10 minutes, 1 hour or 24 hours after training. Ants
 tested at 10 minutes were not tested at any other time points, but all
 ants tested at 1 hour were also tested at 24 hours. After testing, ants
 were briefly presented with the sugar reward to assess whether they were
 still motivated to feed. Ants that did not feed after testing were
 excluded from analysis. All ants were kept harnessed until the last test
 ended. Ants harnessed overnight for 24 hour testing were kept in a dark
 and humid environment.  (d) Scoring and statistical
 analysis<br> We scored whether ants performed the maxilla-labium
 extension response (MaLER) during the 10 second presentation of the
 conditioned stimulus (CS) for each training trial and test (see Fernandes
 et al., 2018). All statistical analysis was performed using R (RStudio
 v1.0.143). To analyse whether the propensity to perform MaLER increased
 during training and whether it differed among different training groups,
 we ran a logistic regression model with mixed effects (Bates, 2010), using
 the ‘lme4’ package. Models were implemented using the glmer command
 incorporating fixed (trial number, training type) and random (individual)
 effects (see supplementary materials). We used Barnard’s test (Barnard,
 1989) to compare the proportion of responses in each group during each
 test with the ‘barnard’ package. One-tailed or two-tailed Barnard’s tests
 were applied depending on the specific test (see supplementary materials).
  To determine whether ants responding during testing were also those that
 learnt, we used a classification of ‘learners’ or ‘non-learners’
 established in Fernandes et al. (2018); those individuals that responded
 on 4 or more of the 10 training trials were ‘learners’. Bonferroni
 corrections (Bonferroni, 1936) were applied when appropriate (see
 supplementary materials). <br>