DOI: 10.5061/dryad.8sf7m0cp5
title: Re-emergence and diversification of a specialised antennal lobe morphology in ithomiine butterflies

description: How an organism’s sensory system functions is central to how it navigates
 its environment. The insect olfactory system is a prominent model for
 investigating how ecological factors impact sensory reception and
 processing. Notably, work in Lepidoptera led to the discovery of vastly
 expanded structures, termed macroglomerular complexes (MGCs), within the
 primary olfactory processing centre. MGCs typically process pheromonal
 cues, are usually larger in males, and provide classic examples of how
 variation in the size of neural structures reflects the importance of
 sensory cues. Though prevalent across moths, MGCs were lost during the
 origin of butterflies, consistent with evidence that courtship initiation
 in butterflies is primarily reliant on visual cues, rather than long
 distance chemical signals. However, an MGC was recently described in a
 species of ithomiine butterfly, suggesting that this once lost neural
 adaptation has re-emerged in this tribe. Here, we show that MGC-like
 morphologies are widely distributed across ithomiines, but vary in both
 their structure and prevalence of sexual dimorphism. Based on this
 interspecific variation we suggest that the ithomiine MGC is involved in
 processing both plant and pheromonal cues, which have similarities in
 their chemical constitution, and co-evolved with an increased importance
 of plant derived chemical compounds.
description: <b><i>i)
 Animals</i> </b> Specimens
 were collected in the Estación Científica Yasuní, in the Parque Nacional
 Yasuní, Orellana Province, Ecuador, during November-December 2011, and
 September-October 2012, under permit 0033-FAU-MAE-DPO-PNY and exported
 under permits 001-FAU-MAE-DPO-PNY and 006-EXP-CIEN-FAU-DPO-PNY. Permits
 were obtained from Parque Nacional Yasuní, Ministerio Del Ambiente, La
 Dirección Provincial de Orellana. Species representing 12 genera,
 excluding <i>Godyris</i>, were selected on the basis of
 phylogenetic distribution and available sample size, and represent 8 of
 the 10 ithomiini subtribes (Table S1; Figure 1). These species are:
 <i>Melinaea spp., Mechanitis polymnia, Forbestra olivencia, Methona
 grandior/curvifascia, Ithomia amarilla, Hypothyris anastasia, Napeogenes
 larina, Oleria gunila, Hyposcada illinissa, Callithomia lenea, Pseudoscada
 florula, </i>and<i> Hypoleria Sarepta</i>. To increase
 sample size for <i>Melinaea</i>, which is in an important
 phylogenetic position, we include individuals from <i>M. menophilus,
 mnasias </i>and <i>marsaeus </i>(see Supplementary
 Material for further discussion).<i> </i>Dissection and
 fixation of specimens were performed at the Estación Científica Yasuní.
 Brains were exposed by removing a section of head carapace under
 HEPES-buffered saline (HBS; 150 mM NaCL; 5mM KCL; 5 mM CaCl2; 25 mM
 sucrose; 10mM HEPES; ph 7.4), before being fixed with zinc formaldehyde
 solution (ZnFA; 0.25% [18.4 mM] ZnCl<sub>2</sub>; 0.788%
 [135mM] NaCl; 1.2% [35mM] sucrose; 1% formaldehyde) for 16-20 hours.
 Extraneous head tissue was then removed, and brains were washed three
 times in HBS. Samples were transferred to 80% methanol/20% DMSO for at
 least two hours, then stored in 100% methanol. Samples were kept at room
 temperature until return to the UK, then transferred to
 -20°C. <b><i>ii)
 Immunohistochemistry </i></b> Samples were rehydrated using
 serial Tris buffer-methanol solutions (90%, 70%, 50%, 30% and 0%), each
 for 10 minutes. Brains were then incubated for two hours in
 NGS-PBS<sub>d</sub> (5% Normal Goat Serum, 1% DMSO, 94% 0.1M
 PBS), before being exposed to anti-SYNORF1 (Antibody 3C11; Developmental
 Studies Hybridoma Bank, University of Iowa, Iowa City, IA, RRID:
 AB_2315424; Buchner, 1996) in solution with
 PBS<sub>d</sub>-NGS, at a ratio of [1:30], for 3.5 days at
 4°C. Non-bound antibody was removed by washing with PBS<sub>d
 </sub>(1% DMSO, 99% 0.1M PBS) three times. Goat anti-mouse secondary
 antibody, Cy2-conjugated (Jackson ImmunoResearch; Cat No. 115–225‐146,
 RRID: AB_2307343, West Grove, PA), was then applied at [1:100] in
 PBS<sub>d</sub>-NGS for 2.5 days at 4°C. Samples were imbued
 with glycerol through graded exposure in 0.1M Tris buffer (1%, 2%, 4% each
 for two hours, and 8%, 15%, 30%, 60%, 70%, 80%, each for 1 hour), and full
 dehydrated by washing with 100% ethanol (3 x 30 minutes). Methyl
 Salicylate was then underlaid, and the brains allowed to sink. This was
 repeated twice before transfer to storage vials of methyl
 salicylate. <b><i>iii)
 </i>Confocal imaging and Image Segmentation</b>
 Samples were mounted in
 methyl salicylate held between two cover slips either side of a hole bored
 through an aluminium slide. <i>Mechanitis</i> and
 <i>Ithomina </i>were imaged on a Leica SP5 microscope using a
 10x 0.4NA objective. All other species were scanned on an Olympus IX3-SSU
 using a 10x 0.4NA objective. As we do not compare raw volumes across
 species, the use of different microscopes does not affect our subsequent
 analyses. For each individual, a single stack was taken encompassing the
 whole of one antennal lobe, chosen at random, with a z-step of 1µm between
 each optical section and a <i>x-y</i> resolution of 1024x1024
 pixels. Consistent light detection was ensured by adjusting the laser
 intensity and gain with depth. To correct for the artefactual shortening
 of the z-dimension of images due to the air objective lenses, a correction
 factor of 1.52 was applied to the <i>z</i>-dimension of the
 image stacks (Heinze and Reppert, 2012; Montgomery and Ott, 2015). Image
 segmentation was performed in Amira 5.4.1 (ThermoFisher Scientific; RID:
 SCR_007353). Volumes of evaluated areas were exported using the
 <i>measure statistics </i>tool. Volumes for surface models
 were plotted using the <i>nat </i>R package (Bates et al.
 2020). Putative
 MGCs were identified on the basis of internal fibrous structure and
 location at the base of the antennal nerve, as described in Montgomery and
 Ott (2015). To formally examine the presence of MGs, each individual
 glomerulus was segmented in two focal male ALs for each species. In
 <i>Methona</i> 4 males were analysed in this way to confirm
 the apparent lack of both MGs and MGCs (see results). Males were chosen
 for this initial assessment as the MGs clustered around the base of the
 antennal nerve are larger in <i>Godyris</i> males, and no
 enlarged glomeruli outside this cluster observed in females (Montgomery
 and Ott, 2015), as is also commonly observed across moths (Rospars and
 Hildebrand, 1992; Huetteroth and Schachtner, 2005; El Jundi et al., 2009b;
 Løfaldli et al., 2010; Yan et al. 2019). Three tests were used to evaluate
 whether MGs were present in focal individuals, based on methodologies used
 in previous publications (Montgomery and Ott, 2015; Keubler et al, 2010;
 Kelber et al. 2009). As the method used by Kelber et al (2009) was found
 to be the most conservative (see Supplementary File) we focus on these
 results in the main text. Under this method, a glomerulus is considered to
 be a MG if its volume is greater than the 90th percentile of glomeruli
 volumes plus <i>k</i> times the difference between the
 10<sup>th</sup> and 90<sup>th</sup> percentiles
 (Kuebler et al. 2010). We
 discriminated between MGs and normal glomeruli using a threshold k value
 of 1.5, which defines a moderate outlier (Sachs, 1988). MGs were
 considered to be present if they passed the discrimination threshold in
 both fully segmented males. We note that our definition of a MG is
 dependent upon the volumetric distribution of the other glomeruli, meaning
 that expansion of non-MGC glomeruli may obscure the presence of glomeruli
 expanded to a similar degree as MGs in other genera. We therefore provide
 the glomeruli size distributions for all species (Figures
 S2-14). For
 subsequent evaluations of sexual dimorphism we scanned multiple males and
 females for each species in the same way as described above. In total 178
 individuals were measured for this dataset with an average of 15
 individuals/species. Samples size for each species are provided in Table
 S1, and range from 2-12 males and 4-11 females. In all individuals of both
 sexes we then segmented: i) all glomeruli comprising the putative MGC,
 including both MGs and closely associated ‘satellite’ glomeruli. Satellite
 glomeruli were included where they form a distinct unit of glomeruli with
 the MGs, which are together raised with respect to the anterior surface of
 the remaining glomeruli, and have the fibrous internal structure
 characteristic of MGCs (Montgomery and Ott, 2015); ii) the combined volume
 of all glomeruli; and iii) total antennal lobe volume including glomeruli
 and the internal antennal lobe hub. Data for <i>Godyris
 zavalata</i> were taken from Montgomery and Ott (2015) but images
 were checked for consistency with newly obtained data.