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.