bioRxiv preprint doi: https://doi.org/10.1101/2020.10.13.336206; this version posted October 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Re-emergence and diversification of a specialised antennal lobe morphology 2 in ithomiine butterflies 3 4 Authors: 5 Billy J Morris1*, Antoine Couto1,2, Asli Aydin3, Stephen H Montgomery2*. 6 7 Affiliations: 1 8 Dept. of Zoology, University of Cambridge, Downing Street, Cambridge, CB2 3EJ 9 2 School of Biological Sciences, University of Bristol, 24 Tyndall Avenue, Bristol, BS8 1TQ 10 3 School of Medicine, Koc University, Rumelifeneri Yolu 34450 Sarıyer / Istanbul, Turkey 11 12 * corresponding authors: 13 BJM: [email protected] 14 SHM: [email protected] 15 16 Abstract 17 How an organism’s sensory system functions is central to how it navigates its environment and 18 meets the behavioural challenges associated with survival and reproduction. Comparing sensory 19 systems across species can reveal how facets of behaviour and ecology promote adaptive shifts 20 in the relative importance of certain environmental cues. The insect olfactory system is prominent 21 model for investigating how ecological factors impact sensory reception and processing. Notably 22 work in Lepidoptera led to the discovery of vastly expanded structures, termed a macroglomerular 23 complex (MGC), within the primary olfactory processing centre. These structures typically process 24 pheromonal cues and provide a classic example of how variation in size can influence the 25 functional processing of sensory cues. Though prevalent across moths, the MGC was lost during 26 the early evolution of butterflies, consistent with evidence that courtship initiation in butterflies is 27 primarily reliant upon visual cues, rather than long distance olfactory signals like pheromones. 28 However, a MGC has recently been reported to be present in a species of ithomiine, Godryis 29 zavaleta, suggesting this once lost neural adaptation has re-emerged in this clade. Here, we show 30 that MGC’s, or MGC-like morphologies, are indeed widely distributed across the ithomiine tribe, 31 and vary in both structure and the prevalence of sexual dimorphism. Based on patterns of 32 variation across species with different chemical ecologies, we suggest that this structure is 33 involved in the processing of both plant and pheromonal cues, of interlinked chemical constitution, 34 and has evolved in conjunction with the increased importance and diversification of plant derived 35 chemicals cues in ithomiines. 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.13.336206; this version posted October 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 36 Key words: 37 Neural adaptation, chemical ecology, Ithomiini, olfaction, pheromones, sexual signalling. 38 39 Introduction 40 An organism’s sensory system is its interface with the rest of the world, the link between its internal 41 and external environments. The manner in which sensory systems vary can reveal how different 42 species are attuned to different cues, the association between cues and behaviour, and how 43 behavioural variation maps to the evolution of sensory systems. Lepidopterans have often been 44 used as models to investigate how ecological variability affects the evolution of olfactory systems 45 (Carlsson et al. 2011; Bisch-Knaden et al. 2012; Carlsson et al. 2013; Namiki et al. 2014; van Dijk 46 et al. 2017), and how the central brain processes olfactory information (Kanzaki et al. 1989; Anton 47 and Hansson, 1994; Tabuchi et al. 2013). This includes classic work characterising pheromones, 48 the olfactory response to these chemical cues, and the manner in which the pheromone 49 processing system evolves (Butenandt et al. 1959; Klun and Maini, 1979; Namiki et al. 2014). 50 Studies on lepidopteran sensory systems have provided crucial insights into how sensory systems 51 function, how separate strands of information are processed and integrated within the brain, and 52 the relationship between sensory systems and ecological variables (Couto et al. 2020). 53 As in all insects, the primary olfactory processing structure within the lepidopteran brain is 54 the antennal lobe (AL). The AL is composed of a collection of functional and morphological units, 55 termed glomeruli. Each glomerulus is a synapse dense region composed of the axon terminals of 56 antennal sensory neurons that typically express the same olfactory receptor (Vosshall et al. 2000), 57 local interneurons that refine the olfactory message, and projection neurons that convey 58 information to higher brain centres. Odorants elicit activity across a range of olfactory receptors, 59 and associated glomeruli, encoding the odorant identity through the combinatorial activation of 60 glomeruli (Joerges et al. 1997; Galizia et al. 1999; Carlsson et al. 2002; Wang et al. 2003; Hallem 61 and Carlson, 2006; Zube et al. 2007). Despite their ecological diversity, within Lepidoptera the 62 antennal lobe is relatively consistent in its structure, being made up of ~60-80 glomeruli (Rospars, 63 1983; Berg et al. 2002; Kazawa et al. 2009; Heinze and Reppert, 2012; Montgomery and Ott, 64 2015; Montgomery et al. 2016; Zhao et al. 2016). However, in moths a prominent morphologically 65 distinct sub-cluster of glomeruli occur at the base of the antennal nerve (Bretschneider, 1924; 66 Matsumoto and Hildebrand, 1981; Koontz and Schneider, 1987). This glomerular cluster is termed 67 a Macroglomerlar complex (MGC) and is composed of enlarged, ‘macro’ glomeruli (MG), and 68 smaller, associated glomeruli, termed ‘satellite’ glomeruli. These glomeruli often display an 69 extreme degree of sexual dimorphism, being vastly enlarged in males relative to females 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.13.336206; this version posted October 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 70 (Matsumoto and Hildebrand, 1981; Koontz and Schneider, 1987). Though first identified in moths, 71 MGs/MGCs have subsequently been observed in a diverse range of insects, including in 72 Blattodea, Diptera, Hymenoptera, and Lepidoptera (Chambille et al. 1980; Couto et al. 2016; 73 Kelber et al. 2009; Ibba et al. 2010; Keubler et al. 2010). MGCs are typically involved in processing 74 pheromonal cues detected by the antennal sensilla, where their corresponding olfactory receptors 75 are highly expressed in a greater number of sensory neurons, providing heightened sensitivity 76 (Warner et al. 2007; Miura et al. 2009). MGs responsive to host plant related cues have also been 77 reported (Ibba et al. 2010), suggesting they reflect an efficient way of increasing sensitivity to 78 biologically important odours to each species. MGs are therefore classic examples of how 79 neuropil size reflects functional performance, as variation in their volume is generally associated 80 with variation in sensitivity to their corresponding odour (Gronenberg and Hölldobler, 1999; El 81 Jundi et al. 2009b; Warner et al. 2007; Miura et al. 2009). Furthermore, MGC structure and 82 composition is variable across closely related species, suggesting they may co-evolve adaptively 83 with species specific ecological and behavioural traits (Kondoh et al. 2003; Namiki et al. 2014; 84 Bastin et al. 2018). 85 While MGCs are ubiquitous in moths (Rospars and Hildebrand, 2000; Huetteroth and 86 Schachtner, 2005; El Jundi et al., 2009b; Løfaldli et al., 2010; Yan et al. 2019), including diurnal 87 species (Stöckl et al. 2016), they are absent in several phylogenetically disparate butterflies 88 (Rospars, 1983; Carlson et al. 2011; Heinze and Reppert, 2012; Montgomery et al. 2016) 89 suggesting they were lost at the origin of Papilionoidea. This has been interpreted as reflecting 90 an increased reliance on visual cues, and the decreased importance of long-distance chemical 91 signalling in butterfly mating behaviours (Rospars, 1983; Rutowski, 1991; Andersson et al. 2007). 92 However, this view is being revisited. Evidence is accumulating that pheromone cues function in 93 interspecific discrimination, sexual attraction and discrimination, and expediate female 94 acceptance in courtship in a range of butterflies (Andersson et al. 2007; Constanzo and Monteiro, 95 2007; Schulz et al. 2007; Mérot et al. 2015; Chengzhe et al. 2017; Darragh et al. 2017). 96 One diverse tribe of diurnal butterflies with particular reliance on olfactory cues are the 97 Ithomiini. Ithomiines utilise derivatives of pyrrolizidine alkaloids (PAs) for both chemical defence 98 and intraspecific communication (Pliske, 1975, Pliske et al. 1976; Brown, 1984). PAs are 99 sequestered from particular species of plants at the adult stage, with males being significantly 100 more attracted and motivated by these resources (Pliske, 1975; Brown 1984). Males provision 101 eggs with PAs through the spermatophore, providing chemical protection to the egg and larvae 102 (Brown 1984). PA derived pheromones are secreted from ‘hair pencils’, specialised, elongated 103 cells found on the dorsal surface of the androconial gland on the forewing (Schulz et al.1988). 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.13.336206; this version posted October 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 104 The expression of PA-derived pheromones is believed to represent an honest signal of male 105 quality and facilitates mating receptivity in females (Boppré, 1978).