Introduction

Insects have exploited chemical signaling as their primary communication mode (Greenfield 2002; Missbach et al. 2014). Particularly cuticular hydrocarbons (CHCs), nonpolar lipids coating the epicuticle of terrestrial insects, have consistently been demonstrated as pivotal signals and cues in a wide variety of chemical communication systems (Blomquist and Bagnères 2010; Blomquist and Ginzel 2021). Predominantly, CHCs have been shown to be major signaling molecules for nestmate recognition in eusocial taxa (e.g. Leonhardt et al. 2016; Sprenger and Menzel 2020) and for sexual and species-specific signaling mechanisms in solitary taxa (e.g. Chung and Carroll 2015; Shahandeh et al. 2018). It has been suggested that chemical profiles in eusocial insect societies, with their multiple castes, task allocations and collective processes, display a higher degree of complexity than in solitary species (Linksvayer 2015; Korb and Thorne 2017; Kronauer and Libbrecht 2018; Holland and Bloch 2020). However, there is no consensus as to how to assess, quantify, and compare the degree of CHC profile complexity across different species (Friedman et al. 2020; Holland and Bloch 2020). Chemical complexity in CHC profiles has previously been assessed as the total number of compounds of a given type, or total ratio of structurally more complex CHC compounds (i.e., unsaturated and methyl-branched CHCs) versus less complex compounds (i.e.,straight-chain CHCs) (Martin and Drijfhout 2009; Kather and Martin 2015). Taking this approach, Kather and Martin (2015) did not find any correlation between CHC diversity and social complexity in a meta-study comparing chemical profiles in eusocial and solitary Hymenopteran species.
Like the Hymenoptera (ants, bees, wasps, sawflies), the order Blattodea encompasses all known levels of social complexity, from solitary cockroaches to obligately eusocial termites. Particularly in termites, which generally lack well-developed eyes, chemical signaling has been repeatedly demonstrated to be a wide-spread and dominant form of communication (Van der Meer et al. 1999; Bagnères and Hanus 2015). In this context, CHCs have been particularly well investigated as fundamental signaling cues for caste differentiation, nestmate recognition and reproductive status conveyance in termites (Liebig et al. 2009; Weil et al. 2009; Hoffmann et al. 2014). But in solitary cockroaches as well, CHCs appear to carry out diverse signaling functions, such as kin recognition and aggregation (Rivault et al. 1998; Lihoreau and Rivault 2008; Hamilton et al. 2019). To the best of our knowledge, no studies have yet attempted to directly compare CHC diversity across different levels of social complexity within the order Blattodea. In the present study, we compare levels of complexity between CHC profiles of representative solitary and social species within the order Blattodea. Through the availability of whole-genome transcriptomes for our selected study species, we additionally explored CHC biosynthesis gene transcript diversity and correlate it with the different levels of social complexity as well as the respective CHC compound classes they are predominantly associated with.
Our cockroach study species are Blatta orientalis (Blattodea: Blattidae) and Blattella germanica (Blattodea: Ectobiidae). The former is known as one of the most common cockroach pest species in temperate regions around the world (Thoms and Robinson 1986, 1987; Edwards and Short 1993), whereas the latter is well-established in CHC-based chemical communication research (Gu et al. 1995; Rivault et al. 1998; Fan et al. 2003; Pei et al. 2019). Within the termites, although all species are considered eusocial, the level of organization and social complexity varies across different termite societies in terms of colony size, worker sterility and morphological differentiation of castes (Abe 1987; Thorne 1997; Korb and Hartfelder 2008; Korb et al. 2015). One piece life type (OPT) or single-site termite species have a low social complexity with small colonies and totipotent workers, spending their entire lives nesting and feeding within the same enclosed, wood-based habitat (Noirot 1970; Shellman-Reeve 1997; Korb and Thorne 2017). They display an exceptionally flexible caste development, with larval offspring retaining the capability to differentiate into reproductives, alates or soldiers well into their late instar stages (Noirot 1985b; Korb and Hartfelder 2008). This pattern is widely considered to be the ancestral form and is characterized by a low to intermediate form of social complexity (Noirot and Pasteels 1987, 1988; Legendre et al. 2008). Low social complexity OPT termites are represented in our study by the two species Kalotermes flavicollis and Neotermes castaneus(Blattodea: Kalotermitidae). Separate life type (ST) or central-site termite species divide their nesting place from their multiple food sources and are thus characterized by foraging (Noirot 1970; Abe 1987; Shellman-Reeve 1997). As opposed to OPT termites, ST termites are more constrained in their development due to an early instar separation into a wingless (apterous) line that can further differentiate into permanently sterile soldiers and workers and a nymphal line that eventually develops into sexual alates (Noirot 1985a; Korb and Hartfelder 2008; Roisin and Korb 2010). This pattern characterizes the most socially complex termite species which can reach much larger and more differentiated colonies than OPT termites (Noirot and Pasteels 1987, 1988; Legendre et al. 2008). Reticulitermes flavipes andCoptotermes formosanus (Blattodea: Rhinotermitidae) as well asMastotermes darwiniensis (Blattodea: Mastotermitidae) represent ST termites in our study, whereas the latter constitutes a particularly interesting case: The species M. darwiniensis is the only extant member of the family Mastotermitidae and phylogenetically represents the most basal termite lineage. However, this species displays all characteristics of ST termites with large colonies, constrained developmental pathways and a true worker caste (Inward et al. 2007b; Krishna et al. 2013). Since the low social complexity OPT has been widely hypothesized to be the ancestral termite state, the clear ST pattern of M. darwiniensis as basal and most ancient extant termite lineage represents an unresolved and frequently debated conundrum (Inward et al. 2007a; Korb and Thorne 2017; Chouvenc et al. 2021).
We tested the central hypothesis that chemical and social complexity are correlated, and that, concordantly, the genetic repertoire for CHC biosynthesis gene transcripts increases with the level of social complexity. We focused on structurally complex CHC compounds (unsaturated and methyl-branched) and the candidate genes that potentially play a role in their biosynthesis and variation (mostly desaturases and microsomal fatty acid synthases, see Fig. 1 and Holze et al., 2021). Moreover, we constructed a chemical dendrogram based on CHC divergence, compared it to the molecular phylogeny of our study species, and correlated CHC biosynthesis gene transcript counts with the respective CHC compound counts per analyzed species.