Highlights d A phylogenetic tree of termites is reconstructed using 4,065 orthologous genes d Fungiculture in Macrotermitinae postdates the loss of gut protists d Sphaerotermes and Macrotermitinae descended from a common comb-building ancestor d Gut protists were probably replaced by gut bacteria in the ancestor of Termitidae

Background Termites primarily feed on lignocellulose or soil in association with specific gut microbes. The functioning of the termite gut microbiota is partly understood in a handful of wood-feeding pest species but remains largely unknown in other taxa. We intend to fill this gap and provide a global understanding of the functional evolution of termite gut microbiota. Results We sequenced the gut metagenomes of 145 samples representative of the termite diversity. We show that the prokaryotic fraction of the gut microbiota of all termites possesses similar genes for carbohydrate and nitrogen metabolisms, in proportions varying with termite phylogenetic position and diet. The presence of a conserved set of gut prokaryotic genes implies that essential nutritional functions were present in the ancestor of modern termites. Furthermore, the abundance of these genes largely correlated with the host phylogeny. Finally, we found that the adaptation to a diet of soil by some termite lineages was accompanied by a change in the stoichiometry of genes involved in important nutritional functions rather than by the acquisition of new genes and pathways. Conclusions Our results reveal that the composition and function of termite gut prokaryotic communities have been remarkably conserved since termites first appeared ~ 150 million years ago. Therefore, the “world’s smallest bioreactor” has been operating as a multipartite symbiosis composed of termites, archaea, bacteria, and cellulolytic flagellates since its inception.

For sexual communication, moths primarily use blends of fatty acid derivatives containing one or more double bonds in various positions and configurations, called sex pheromones (SPs). To study the molecular basis of novel SP component (SPC) acquisition, we used the tobacco hornworm (Manduca sexta), which uses a blend of mono-, di-, and uncommon triunsaturated fatty acid (3UFA) derivatives as SP. We identified pheromone-biosynthetic fatty acid desaturases (FADs) MsexD3, MsexD5, and MsexD6 abundantly expressed in the M. sexta female pheromone gland. Their functional characterization and in vivo application of FAD substrates indicated that MsexD3 and MsexD5 biosynthesize 3UFAs via E/Z14 desaturation from diunsaturated fatty acids produced by previously characterized Z11-desaturase/conjugase MsexD2. Site-directed mutagenesis of sequentially highly similar MsexD3 and MsexD2 demonstrated that swapping of a single amino acid in the fatty acyl substrate binding tunnel introduces E/Z14-desaturase specificity to mutated MsexD2. Reconstruction of FAD gene phylogeny indicates that MsexD3 was recruited for biosynthesis of 3UFA SPCs in M. sexta lineage via gene duplication and neofunctionalization, whereas MsexD5 representing an alternative 3UFA-producing FAD has been acquired via activation of a presumably inactive ancestral MsexD5. Our results demonstrate that a change as small as a single amino acid substitution in a FAD enzyme might result in the acquisition of new SP compounds.fatty acid desaturase | Manduca sexta | sex pheromone biosynthesis | pheromone evolution | substrate specificity S ex pheromones (SPs) are a diverse group of chemical compounds that are central to mate-finding behavior in insects (1). Variation in SP composition between closely related species and among populations is well documented. Despite this variation, SPs are presumed to be under strong stabilizing selection, and thus the genetic mechanisms driving SP diversification represented an enigma (2). Research on SPs in moths (Insecta: Lepidoptera) helped establish the hypothesis of asymmetric tracking as a major driving force in SP diversification. In this scenario, abrupt changes in female SP composition via a shift in component ratio or the inclusion or loss of a component result in a distinct SP that attracts males with more broadly or differentially tuned SP preference (3). Assortative mating, the preferential mating of females producing a novel SP with males attracted to this SP, restricts gene flow between subpopulations with differing SP compositions. This can ultimately lead to speciation and fixation of novel communication channels (4). Work in insect models such as wasps (5), fruit flies (6), and especially moths (7-9) is helping uncover the genetic basis of SP diversification.In the majority of moth species, females use species-specific mixtures of SP components (SPCs) consisting of volatile fatty acid (FA) derivatives to attract conspecific males at long range. These SPCs are predominantly long-chain aliphatic (C12-C18) acetates, alcoh...