DOI: 10.5061/dryad.9w0vt4bdh
title: Caterpillar polarisation vision: Histological methods and Rcodes for behavioural analyses

description: Processionary caterpillars of Thaumetopoea pityocampa (in Europe) and
 Ochrogaster lunifer (in Australia) (Lepidoptera: Notodontidae) form single
 files of larvae crawling head-to-tail when moving to feeding and pupation
 sites. We investigated if the processions are guided by polarisation
 vision. The heading orientation of processions could be manipulated with
 linear polarising filters held above the leading caterpillar. Exposure to
 changes in the angle of polarisation around the caterpillar resulted in
 orthogonal changes in heading angles. Anatomical analysis indicated
 specialisations for polarisation vision of stemma I in both species.
 Stemma I has a rhabdom with orthogonal and aligned microvilli, and an
 opaque and rugged surface; which are optimisations for skylight
 polarisation vision, similar to the dorsal rim of adult insects. Stemmata
 II-VI have a smooth and shiny surface and lobed rhabdoms with
 non-orthogonal and non-aligned microvilli; thus, optimised for general
 vision with minimal polarisation sensitivity. Behavioural and anatomical
 evidence reveal that polarised light cues are important for larval
 orientation and can be robustly detected with a simple visual system.
description: <strong>Materials and Methods</strong>
 Behavioural analyses In August 2018 and 2020, outdoor
 experiments with first instar (L1) <em>T. pityocampa</em>
 larvae were conducted on sunny days between 0930 – 1130 h (GMT +2) at
 Tregnago, Veneto, Italy (45°51’ N, 11°17’ E). A sheet of 50
 cm<sup>2</sup> white paper was used as the experimental arena
 where 10 first instars at a time were released in the middle of the sheet
 and behavioural observations were made; N = 58 observations (31 single
 larvae (singletons) and 29 two or more larvae (processions)). After
 release, the larvae clustered for a few minutes, then formed processions
 or travelled as a singleton in various orientations. Four treatments were
 applied to the processions/singletons after the larva(e) established a
 course. A flexible 25 cm<sup>2</sup> linear polarising filter
 (PF) for visible light (XP42HE-40, ITOS, Mainz, Germany) was bent into a
 tunnel and held above the procession leader or singleton (Supplementary
 Fig. S1). The PF that filtered the light incident to both sides of the
 head was held either (1) ‘horizontally’ or (2) ‘vertically’ by rotating
 the filter 90°; so the horizontally or vertically polarised light was
 transmitted at low elevations, respectively, creating two orthogonal
 polarisation patterns around the larvae. After application, the larvae
 proceeded under (3) unobstructed sky without a filter. The PF created
 shade (transmission to unpolarised light 40%), and additionally decreased
 the incident light by max. 30%, depending on the angle with respect to the
 polarised skylight [21]. As the stemmata are non-image forming organs with
 large fields of view, the possible intensity artefact slightly affected
 the total, but not the differential signal in the orthogonal photoreceptor
 pairs. Thus, (4) control experiments were performed using a 45% neutral
 density filter (NDF) (Lee 298 and 209 combined; Lee Filters, Hampshire,
 UK), in place of PF. Each treatment lasted for 2 min, and the larval
 orientation was recorded 20 s after commencement (Fig. 1). The larvae were
 changed after every trial. The same experiments were conducted
 on final 8th instar (L8) <em>O. lunifer</em> (N = 12
 processions) and 5th instar (L5) <em>T. pityocampa</em> (N = 7
 processions) larvae in their natural habitat without the arena.
 <em>Ochrogaster lunifer</em> were observed in late March 2019
 at 0600 – 1200 h (GMT+10) at The University of Queensland, Gatton campus,
 Queensland, Australia (‑27°56’ S, 152°34’ E). Summer feeding
 <em>T. pityocampa</em>  [22] were observed in September 2019
 at Leiria, Portugal (39°31’ N, 9°07’ W) at 0800 – 1300 h
 (GMT+1). Morphological analyses Preparation of stemmata for
 scanning electron (SEM), light (LM) and transmission electron (TEM)
 microscopy was performed as described previously [23]. Details can be
 found online [24]. Statistical analyses All analyses were performed using R
 Studio version 1.1.419 and an alpha value of P &lt; 0.05 was taken as
 statistically significant. In some trials, the PF was applied and removed
 repeatedly on the same procession/singleton. Before pooling the data for
 analyses, we tested if the orientation of processions was affected by
 previous exposure to the PF. The Wallraff Test of Angular Distances was
 performed using the RStudio software package ‘circular’ [25] on all
 processions/singletons. There were no significant differences (all P
 &gt; 0.1), therefore, the data were used as independent for each
 treatment. To determine if the procession/singleton reacted to the
 treatments, the angular difference was calculated by subtracting the
 initial orientation of travel from the manipulated orientation after
 PF/NDF exposure/removal. Summarised angular differences of each group
 after the treatments were graphed as circular plots with a kernel density
 estimation (Fig. 1). The angular difference was given a score of 0 or 1
 according to the difference being less (no change) or greater than 22.5°
 (change; 22.5° is the minimal cardinal value on the compass). Circular
 Logistics Regression Model (CLRM) for Binomial Data [26] was applied on
 the angular difference using the variables: Treatment, Azimuth angle
 (degrees), Procession/singleton ID, Number of larvae in the procession and
 Starting orientation. The models were reduced to the model of best fit by
 removing non-significant covariables and by lowering the Akaike’s
 information criterion value. Environmental temperatures of the study sites
 were collected, but not used as a variable because of collinearity
 (r &gt; 0.70) with the Azimuth angle. Details of R codes used can be
 found online [24].
description: For further details on Morphological analyses and Statistical
 analyses, download the Dryad datasets.