Discussion
CHC divergence and variation in relation to social
complexity
We compared chemical and associated transcriptomic complexity between
Blattodea species with increasing levels of social organization.
Overall, we did not find a consistent correlative pattern of CHC-based
chemical complexity paralleling the different levels of social
complexity in our representative Blattodea species. These results point
to large categorical differences in CHC profiles across species, which
appear to be independent from their level of social organization.
CHC divergence based on average chemical distances between the species
neither reflects the different levels of complexity nor the phylogenetic
divergence within the Blattodea (Fig. 3). M. darwiniensis , the
most basal termite lineage (Inward et al. 2007b; Krishna et al. 2013)
chemically clusters together with both a high (R. flavipes ) and a
low (N. castaneus ) social complexity termite, and the solitary
cockroach B. orientalis clusters with both a high (C.
formosanus ) and a low (K. flavicollis ) social complexity
termite. It has been hypothesized that chemical divergence clearly
differing from an established molecular phylogeny indicates selection on
chemical profiles for different functions overriding their phylogenetic
information (Marten et al. 2009; Buellesbach et al. 2013). However, our
chemical divergence does not display any pattern congruent with the
social hierarchy of our study species, rendering any assumptions on
selection for CHC functions reflective of the species’ respective social
complexity highly unlikely. Concerning counts of gene transcripts
stemming from orthologs of CHC biosynthesis genes from other insect
species, these do neither quantitatively correlate with higher levels of
social complexity nor with the total number of CHC compounds detected in
each of our study species.
Future work could include more species as transcriptomes, genomes and
chemical profiles become available, and utilize scalable frameworks such
as phylogenetically-contrasted regression and Bayesian ancestral state
reconstruction models (Simon et al. 2019). Additionally, caste-specific
CHC variation in eusocial taxa could be taken into account in future
studies as well, potentially adding another layer of complexity, despite
the accompanying issues for direct comparability with solitary taxa.
CHC biosynthesis gene transcript variation across the
studied Blattodea
species
Acetyl-CoA carboxylase (ACC) catalyzes the first and rate-limiting step
in CHC biosynthesis (Barber et al. 2005, see Fig. 1). In each of our
analyzed cockroach and termite species, we found several distinct ACC
transcripts (ranging from 3 in C. formosanus to 8 in B.
germanica ) based on orthology to the Drosophila gene (Tab. 1).
This rich abundance of ACC transcripts strengthens the argument for the
universality of ACC as fundamental catalyst for the first step in CHC
biosynthesis (see also Fig. 5).
For FAS genes, seven of them had already been identified inBlatella germanica , with five showing a significant effect on CHC
compound quantities upon knockdown (Pei et al. 2019). In our analyzed
transcriptomes, we were able to detect transcripts with strong orthology
to two of these five FAS genes (BgFas 4 and 6). Transcripts with
orthology to BgFas4 were detectable across all seven species,
whereas BgFas6 transcripts were most abundant in the two
cockroaches and only in three of the five analyzed termites (Tab. 1).
Generally, it has been hypothesized that two types of FAS, cytosolic and
microsomal, differentially impact CHC biosynthesis, with the former
mainly governing non-methylated, straight-chain CHCs, and the latter
being more specific for methylated CHCs (Chung et al. 2014;
Wicker-Thomas et al. 2015, Fig. 1). Since we could not detect any
transcript copies of BgFas6 in both M. darwiniensis andN. castaneus , the two species with the lowest amounts of
methyl-branched alkanes (Fig. 2), it is possible that the BgFas6transcripts stem from a microsomal FAS associated with the production of
methyl-branched alkanes. Intriguingly, for N. castaneus , in
addition to showing both the lowest number and proportion of
methyl-branched alkanes, it also shows the lowest transcript copy number
of FASN2 orthologs, an oenocyte-specifically expressedDrosophila gene with a strong effect on methyl-branched CHCs and
thus speculated to be microsomal (Chung et al. 2014; Wicker-Thomas et
al. 2015).
Unsaturated compounds, whose biosynthesis is crucially dependent on
desaturases (Wicker-Thomas et al. 1997; Coyne et al. 1999; Dallerac et
al. 2000), occur in each of our studied Blattodea species, most
abundantly in R. flavipes , M. darwiniensis and N.
castaneus , intermediately in B. orientalis and B.
germanica , but only in traces in K. flavicollis and C.
formosanus (Fig. 2). This partially reflects the respective numbers of
transcripts orthologous to the genes desat1 and desat2 ,
with the former being most abundantly represented in M.
darwiniensis and N. castaneus , and the latter in R.
flavipes (Tab. 1).
Genes coding for P450 Decarbonylases of the gene subfamily CYP4G have
been shown to govern the final steps in CHC biosynthesis and have thus
been suggested to be stable, highly conserved and particularly vital
elements in this pathway (Feyereisen 2020; Holze et al. 2021, Fig. 1).
Concordantly, at least one Cyp4g gene could be identified in all
insect genomes screened to date (Qiu et al. 2012; Feyereisen 2020). We
found transcripts orthologous to the Drosophila geneCyp4g1 as well as the migratory locust Locusta migratoriagene LmCYP4G102 in all our tested species (Tab. 1). However,
their numbers vary largely with no apparent consistent pattern, hinting
at more transcriptomic variety for these vital CHC biosynthesis elements
than previously assumed.
The assessment of transcript counts as approximation of the actual
genomic repertoire for CHC biosynthesis genes naturally has its limits
and will remain speculative until targeted knockdown studies confirm the
actual functions of the transcripts as well as their underlying genes.
However, analyzing the abundance of unique transcripts per ortholog
allows valuable insights into functional diversity potentially exceeding
the information contained within whole-genome sequences (He et al. 2021;
Sprenger et al. 2021). Correlating transcriptomic diversity directly
with CHC profile variation has been attempted surprisingly rarely
despite its potential to approximate the genetic control of CHC
variation more accurately, as it has repeatedly been shown that the same
genotype can produce different CHC profiles (Holze et al. 2021; Sprenger
et al. 2021). Thus, our findings constitute promising first steps for
future studies with the potential to corroborate our transcriptomic
assessment with target gene counts once further genomic resources become
available.
CHC-based communication mechanisms and outlook for future
studies
It has long been hypothesized that the complexity of CHC profiles
reflects the complexity of chemically encoded communication mechanisms
necessary to maintain more socially complex insect societies (Korb and
Thorne 2017; Kronauer and Libbrecht 2018; Holland and Bloch 2020).
However, how CHC profiles actually encode biologically relevant
information such as nestmate affiliation or task allocation has remained
largely elusive so far (Buellesbach et al. 2018; Menzel et al. 2019;
Heggeseth et al. 2020). Further elucidation on the exact encoding
mechanisms in CHC profiles and which compound combinations actually
convey chemical information will be instrumental in gaining a better
understanding on how insect populations and societies are maintained at
different levels of social complexity. Furthermore, although insects
generally synthesize the majority of the components in their CHC
profiles themselves (Nelson and Blomquist, 1995; Blomquist and Bagnères,
2010), several studies have demonstrated the impact of several biotic
and abiotic factors such as diet, habitat and microbiome on CHC profiles
as well (Fedina et al. 2012; Rajpurohit et al. 2017; Teseo et al. 2019).
Thus, disentangling these factors from the conserved CHC profile
functionalities represents an additional challenge in future studies
that will nevertheless be indispensable to fully comprehend and explore
CHC-mediated communication mechanisms.
Future studies should also consider the occurrence of very-long chained
CHC compounds of up to C58 in Blattodea surface profiles as recently
demonstrated with non-standard analytical methods (Golian et al. 2022).
However, neither exact compound quantifications nor identifications
(e.g., discrimination between n -alkanes and methyl-branch
alkanes) are so far possible in this higher chain length range extending
beyond the CHC compounds traditionally accessed and identified through
gas-chromatographic separation (Schnapp et al. 2016; Bien et al. 2019).
Therefore, to include very long-chain CHCs in future analyses, novel
methods are necessary to reliably assess their exact quantities and
compound classes. Moreover, despite CHCs clearly constituting the
dominant, most investigated compounds in chemical signaling (Blomquist
and Bagnères 2010; Chung and Carroll 2015) and have been implied to have
convergently evolved, unifying communication modalities in eusocial
insects (Leonhardt et al. 2016; Funaro et al. 2018), we cannot exclude
the additional potential of non-CHC compounds also used in signaling
which might reflect social complexity (e.g. Hanus et al. 2010; Smith et
al. 2016; Steitz et al. 2019). Lastly, CHC metabolic networks could be
amenable to various kinds of more elaborate complexity analyses such as
metabolic network pathfinding (Kim et al. 2017), identification of
critical connectors (Kim et al. 2019), determination of topological
characteristics (Goryashko et al. 2019), and flux balance analysis
(Beguerisse-Díaz et al. 2018). These types of complexity analyses have,
to our knowledge, not been attempted so far in this context at all and
might largely aid in obtaining a more holistic correlative view on
complexity on a chemical, genetic and social level.