Eusociality, one of the major evolutionary transitions [1
], refers to a social organization defined by cooperative brood care, overlapping generations, and a division of labour into reproductive and non-reproductive castes [2
]. Eusociality has appeared independently across different groups in the tree of life, but is mainly known in arthropods, particularly in hymenopterans (ants, bees, and wasps) and in termites, as well as in crustaceans (snapping shrimp). The earliest appearance of eusociality, in termites, is supposedly older than 150 Ma (reviewed in [3
]). The emergence of eusociality through natural selection can be explained by kin selection theory [4
] coupled with high relatedness enhanced by monogamy [5
]. Eusocial transitions led to complex innovations, such as cooperative behaviour with task specialization, complex communication, and increased fertility and longevity (breaking the fecundity/longevity trade-off) [6
]. These adaptations led to viewing eusocial colonies as a “super-organism”, similar to the evolution of multicellular organisms, where the reproductive caste is seen as the germline and the non-reproductive caste as the soma, specialising in specific tasks [7
]. In addition, eusociality led to a change of population structure, reducing the effective population size and increasing the generation time effect, which impacts major factors of evolutionary processes [9
]. Indeed, a reduced effective population size weakens selection strength and strengthens the effect of genetic drift [12
]; a longer generation time leads to an increased mutation rate [10
]. However, the molecular basis underlying such complex adaptations remains elusive.
Some of the most puzzling genomic innovations are triggered by Transposable Elements (TEs), mobile genetic elements that were once seen only as genomic parasites (reviewed in [13
]). While most TEs reach fixation within genomes through genetic drift [14
], TEs can promote the ability of populations to adapt to rapidly changing environments (reviewed in [15
]). Indeed, TE insertions can significantly alter phenotypes, contrary to point mutations. There are five main different mechanisms through which TEs can drive adaptive evolution: domestication/exaptation, where TEs give rise to new proteins; the change of gene expression due to nearby TE insertion; gene duplication with TEs inserting transcribed genes into other genome locations; fast evolving genomic regions; chromosomal rearrangements after ectopic recombination, resulting in the pairing of similar TE copies present in different chromosome regions (reviewed in [15
]). The different mechanisms by which TEs can drive adaptive evolution along with their involvement in adaptive radiation in bats, primates, and lizards [17
] indicate the pivotal role of TEs in species diversification. This is further supported by the fluctuation of TE propagation over evolutionary time with bursts of TE propagation following environmental stress and TE horizontal transmission (reviewed in [16
]). However, the link between the TE-mediated phenotypic innovations driven by a specific mechanism is still missing.
In social bees, a decrease in transposable element diversity and abundance correlates with social complexity. Such a decrease could act positively on reducing kin conflict, thereby increasing relatedness among nest mates and, hence, indirect fitness [21
]. Similarly, ancestral reconstruction of TE abundance and diversity in snapping shrimp indicates a moderate abundance of TEs predates and may have driven eusocial transition [22
]. However, contrary to eusocial bees, eusocial snapping shrimp exhibit higher accumulation of TEs compared to non-eusocial species [22
]. While the reduction of TE abundance could have favoured eusocial transition, eusocial-related demographic changes can lead to TE accumulation [22
]. The reduction of TEs found in eusocial bees might be due to a trade-off between genomic diversity and integrity driven by recombination (diversity) and TE silencing (integrity) found in relation to increasing colony size in honey bees [23
]. To further our understanding of the TE evolutionary dynamics and role in adaptive evolution during eusocial transition, we need to expand our investigation to more clades where eusociality has emerged.
Blattodea includes termites and cockroaches. All termites are eusocial, whereas cockroaches exhibit different social organizations, from solitary to subsocial ways of life [24
]. Given this diversity and the latest advances in their phylogenetic relationships [25
], this group is becoming a great model for comparative evolutionary analyses. Moreover, termites are insects with a major economic and ecological importance, but they remain understudied, especially when compared to Hymenoptera. Yet, they drastically differ from eusocial hymenopterans, being hemimetabolous (no metamorphosis to reach adult stage), and from snapping shrimp, as the reproductive division of labour is based on life stages, where workers are juveniles (nymphs). As eusocial shrimp, termites (but also cockroaches) exhibit high TE genomic content [28
]; however, the TE evolutionary dynamics in these species remains largely unknown.
A study of three termite species and one gregarious cockroach species identified a genomic signature of eusociality in termites [28
]. The presence of transposons in the flanking regions of expanding gene families could have facilitated the transition to eusociality. Indeed, a significant amount of insertions into regions adjacent to expanding gene families was observed in termites and absent in the gregarious cockroach, suggesting that these repeated element insertions would be shared among Isoptera. Gene duplication in eusocial hymenopterans has also been found to underlie eusocial transition [29
]. Another major molecular basis underlying eusocial transition is an increase in gene regulatory potential, which could enable phenotypic plasticity [21
]. A major impact of TE insertions near genes is a change of gene expression, through disruptions of regulatory regions and changes of the methylation state (reviewed in [16
]). Hence, we hypothesised that TEs might have facilitated eusocial transition in termites through enhanced gene regulation.
In order to understand if TE-aided adaptive evolution could have led to shifts of gene regulation during eusocial transition in Blattodea, we categorized and annotated TE abundance and diversity in 2 cockroach and 4 termite genomes and determined gene expression bias in different life stages in those species. First, we investigated TE evolutionary dynamics in Blattodea. By mapping TEs onto the blattodean genomes, we investigated whether TEs insert more frequently in genes with differential gene expression between castes in termites and stages in cockroaches. More precisely, we focused on the queen (reproductive) and worker (sterile) castes in termites, and on nymphs (corresponding to the worker caste of termites) and female adults (corresponding to the queen caste of termites) in cockroaches. Furthermore, we explored if a high rate of TE insertions in genes with differential expression among castes in termites could be linked to functions important for eusocial adaptations. Our results indicated that TE accumulation within genes is driven mostly by selection and that a high rate of TE insertions in termite queen- and worker-biased genes seems to be linked to specific functions associated with eusocial adaptations in each caste.
Investigating the role of TEs in social evolution, our study revealed opposite trends of TE insertions within genes with biased expression in cockroaches and termites. Moreover, contrary to cockroaches, in termites, different TE superfamilies have more frequent insertions within genes according to their expression bias between castes. In cockroaches, TEs inserted more frequently, regardless of their superfamily, in genes with no bias in expression or in genes over-expressed in adult females, while nymph-biased genes showed a reduced number of TE insertion. In termites, on the other hand, TEs inserted more frequently, especially retrotransposons (Gypsy
and LINE), in worker-biased genes (corresponding to nymph-biased genes in cockroaches). This trend could be explained by the differential selection strength on cockroach nymph-biased and termite worker-biased genes, as workers are not under direct selection, since they do not reproduce [67
]. Hence, the reduced selection strength could explain the higher proportion of TE insertions in worker-biased genes in termites. This was confirmed by the highest propensity of TEs to insert within exons of worker-biased genes in termites compared to other DEGs and to cockroaches. In cockroaches, TEs inserted more often in the exons of adult-female-biased genes. However, changes in population structure associated with eusociality did not lead to higher TE insertions within genes in those species, suggesting that weaker selection and genetic drift may not have played a major role in TE insertion within genes. An alternate non-exclusive hypothesis, to explain such differences in TE insertion within genes, would rely on the “superorganism” view. Within the “superorganism” view, queen-biased genes correspond to the germline genes, where TE silencing is predominant [70
], while worker-biased genes correspond to somatic cells, where the deleterious mutagenic impact of TEs should not affect fitness as much as in the germline [72
]. In cockroaches, the negative mutagenic impact of TEs on fitness should be greater in nymph-biased genes than adult-female-biased genes, as deleterious mutations on nymph-biased genes should lead to a drastic reduction in fitness as nymphs are not reproductively active. Furthermore, the lack of a pattern of TE insertions of different TE superfamilies within DEGs in cockroaches and the clear pattern in termites of retrotransposons and DNA transposons being more frequent in worker-biased and in queen-biased genes, respectively, suggest the potential role of specific TE superfamilies during the evolution of termites. Indeed, retrotransposons inserting more often in worker-biased genes may favour their shifts in expression during eusocial transition, as has been previously suggested with LINE retrotransposons favouring gene family expansions during termite evolution [28
]. DNA transposons may also play an adaptive role in eusocial transition by inserting more frequently in queen-biased genes. This result is in favour of the role of TEs in adaptive evolution during eusocial transition in Blattodea.
To understand if TE-aided adaptive evolution could have, at least partly, driven the eusocial transition, notably by facilitating shifts in gene regulation, an essential process in eusocial adaptation [21
], not only the more frequent insertion of TEs in caste-specific genes, but the function of those genes need to be linked with adaptations associated with eusociality.
Our analyses of functional categories enriched among TE-rich genes revealed a large number of TEs inserted in queen-biased genes that could be essential for reproductive functions of queens. Indeed, several functional categories that were enriched in TE-rich queen-biased genes are indirectly (“regulation of growth”, “fat cell differentiation”, and “anatomical structure morphogenesis”) and directly (“multi-multicellular process”, “regulation of reproductive process”, and “meiotic nuclear division”) related to reproduction. Hence, numerous TE insertions within queen-biased genes may have facilitated a change in their expression to enhance egg production. Moreover, a high rate of TE insertions in queen-biased genes is linked to functions that could explain ageing adaptations in termite queens, such as “cellular respiration”, “insulin receptor signalling pathway”, and “fat cell differentiation”. Oxidative stress is one of the major impacts of ageing [6
], and mutations in the cellular respiration pathway could impact the effect of oxidative stress [73
]. Moreover, a relationship between ageing and insulin signalling has been established in several species [74
], notably some termite species [75
]. However, no clear trend of differential gene expression involved in the insulin pathway has been found across eusocial insects in relation to ageing and fecundity [6
]. Nevertheless, TE-rich queen biased genes involved in fat cell differentiation could correspond to the adaptation to reproduction and ageing in termites, as evidence suggests the role of vitellogenesis, primarily produced in fat bodies [77
], in ageing and fecundity adaptation in eusocial insects [6
]. Therefore, the insertion of TEs within genes involved in those functions could have facilitated the adaptation in termite queens, leading to the absence of a trade-off in fecundity/longevity. Another function enriched in TE-rich queen-biased genes that could be linked to eusocial adaptation is immunity (“lymphocyte mediated immunity”, “B cell activation”, “regulation of leukocyte differentiation”, “negative regulation of immune system process”). Indeed, social adaptation is thought to lead to increased threat from parasites, due to the high density of closely related individuals, providing a rich environment for parasites [78
]. Hence, enhanced regulation of immunity in termite queens may be of prime importance to modulate immune response at different colony life stages [79
]. Nevertheless, the eusocial transition in termites is accompanied by a reduction of immune gene diversity and activity [80
], which could explain high TE insertions in those genes that may not be active.
In worker-biased genes, enriched functions of TE-rich genes linked with the regulation of gene expression (“positive regulation of gene expression”, “gene expression”, “RNA metabolic process”, “response to endogenous stimulus”) and behaviour (“behaviour”, “adult locomotory behaviour”, “nervous system process”) could be related to eusocial adaptation. Indeed, behaviour-related genes are likely important for cooperative and diverse tasks undertaken by workers in a termite colony [24
]. Furthermore, the phenotypic plasticity observed in eusocial insects has been hypothesized to have been favoured by enhanced gene regulation [21
]. Hence, the high rate of TE insertion in worker-biased genes linked with gene expression could have facilitated the eusocial transition, enabling shifts and plastic gene expression. Furthermore, high TE insertions in worker-biased genes have been linked to processes involved in interaction with the host, which could be related to symbiotic relationships between termite protozoan to digest cellulose [24
]. Finally, the high rate of TE insertions in nymph-biased genes in cockroaches seems mainly to be linked to development (“tube development”, “cell communication”, and “cell differentiation”) and metabolism (“lipid biosynthetic process”, “regulation of metabolic process”, “positive regulation of molecular function”, and “organophosphate metabolic process”) and may enhance the regulation of gene expression during this life stage [24
The fixation of TEs is mainly driven by genetic drift [14
]; however, the reduced effective population size caused by eusocial adaptation [11
] does not seem to have led to higher TE insertions in termites compared to cockroaches. Moreover, the insertion of TEs seems to be more recent, as TE copies are less divergent from their reference TE, in cockroaches compared to termites. However, the generation time effect, leading to higher mutation rates, expected in eusocial insects [10
], might also lead to higher mutations of TEs in termites. The generation time effect could explain greater sequence divergence among related TEs observed in termites compared to cockroaches. While TE superfamily content varies across species, no clear trend was found of different TE superfamilies among eusocial and gregarious blattodeans.
Our study lays the groundwork for studying the role of TEs in shifts of gene regulation during the transition to eusociality and warrants further investigations on the subject to precisely pinpoint the impact of TEs during eusocial evolution. However, our study suffered from some caveats: (1) the lack of manual curation of TEs ensuring a highly reliable TE database in Blattodea, (2) the lack of standardization in the transcriptomic methodology and the surprisingly low number of DEGs found in P. americana
, rendering our subsequent analyses biased toward B. germanica
results for the gregarious species, and (3) the lack of direct evidence of the TE insertions impact on gene expression among gregarious and eusocial Blattodea, as TE insertion in genes could also lead to exon disruption, exonisation, the creation of a new polyadenilation site, and alternative splicing (reviewed in [16
]). In order to further our understanding, Blattodea genomes are necessary, especially species with various social levels. In our study, we only considered gregarious vs.
eusocial blattodeans, while a great variety of social levels are present in cockroaches, notably with subsocial behaviour in cockroach species or distinct eusocial levels in termites, with lower and higher termites [81
]. In addition, to directly categorize the role of TEs in gene expression shifts during the eusocial transition, functional studies are needed to unravel the impact of DEGs with TEs on eusocial adaptation. Furthermore, follow-up comparative genomic studies are needed to unravel how and on which ortholog genes TEs can lead to a change in gene expression between cockroaches and termites, notably through investigating methylation state or chromatin access, and comparing orthologs with the opposite direction of differential expression between cockroaches and termites. Another important factor to further our understanding would be to include transcriptomes of more life stages, as well as better-quality, tissue-specific, and standardized transcriptomes. To conclude, our study provides the first evidence of TE-aided eusocial adaptation in termites through possible changes of gene expression in different castes.