Termites normally rely on gut symbionts to decompose organic matter but the Macrotermitinae domesticated Termitomyces fungi to produce their own food. This transition was accompanied by a shift in the composition of the gut microbiota, but the complementary roles of these bacteria in the symbiosis have remained enigmatic. We obtained high-quality annotated draft genomes of the termite Macrotermes natalensis, its Termitomyces symbiont, and gut metagenomes from workers, soldiers, and a queen. We show that members from 111 of the 128 known glycoside hydrolase families are represented in the symbiosis, that Termitomyces has the genomic capacity to handle complex carbohydrates, and that worker gut microbes primarily contribute enzymes for final digestion of oligosaccharides. This apparent division of labor is consistent with the Macrotermes gut microbes being most important during the second passage of comb material through the termite gut, after a first gut passage where the crude plant substrate is inoculated with Termitomyces asexual spores so that initial fungal growth and polysaccharide decomposition can proceed with high efficiency. Complex conversion of biomass in termite mounds thus appears to be mainly accomplished by complementary cooperation between a domesticated fungal monoculture and a specialized bacterial community. In sharp contrast, the gut microbiota of the queen had highly reduced plant decomposition potential, suggesting that mature reproductives digest fungal material provided by workers rather than plant substrate.carbohydrate-active enzymes | eusocial | symbioses | cellulose | lignin

Gut microbes play a crucial role in decomposing lignocellulose to fuel termite societies, with protists in the lower termites and prokaryotes in the higher termites providing these services. However, a single basal subfamily of the higher termites, the Macrotermitinae, also domesticated a plant biomass-degrading fungus (Termitomyces), and how this symbiont acquisition has affected the fungus-growing termite gut microbiota has remained unclear. The objective of our study was to compare the intestinal bacterial communities of five genera (nine species) of fungus-growing termites to establish whether or not an ancestral core microbiota has been maintained and characterizes extant lineages. Using 454-pyrosequencing of the 16S rRNA gene, we show that gut communities have representatives of 26 bacterial phyla and are dominated by Firmicutes, Bacteroidetes, Spirochaetes, Proteobacteria and Synergistetes. A set of 42 genus-level taxa was present in all termite species and accounted for 56-68% of the species-specific reads. Gut communities of termites from the same genus were more similar than distantly related species, suggesting that phylogenetic ancestry matters, possibly in connection with specific termite genus-level ecological niches. Finally, we show that gut communities of fungus-growing termites are similar to cockroaches, both at the bacterial phylum level and in a comparison of the core Macrotermitinae taxa abundances with representative cockroach, lower termite and higher nonfungus-growing termites. These results suggest that the obligate association with Termitomyces has forced the bacterial gut communities of the fungus-growing termites towards a relatively uniform composition with higher similarity to their omnivorous relatives than to more closely related termites.

Stress-adaptive cell plasticity in target tissues and cells for plant biomass growth is important for yield stability. In vitro systems with reproducible cell plasticity can help to identify relevant metabolic and molecular events during early cell reprogramming. In carrot, regulation of the central root meristem is a critical target for yield-determining secondary growth. Calorespirometry, a tool previously identified as promising for predictive growth phenotyping has been applied to measure the respiration rate in carrot meristem. In a carrot primary culture system (PCS), this tool allowed identifying an early peak related with structural biomass formation during lag phase of growth, around the 4th day of culture. In the present study, we report a dynamic and correlated expression of carrot AOX genes (DcAOX1 and DcAOX2a) during PCS lag phase and during exponential growth. Both genes showed an increase in transcript levels until 36 h after explant inoculation, and a subsequent down-regulation, before the initiation of exponential growth. In PCS growing at two different temperatures (21°C and 28°C), DcAOX1 was also found to be more expressed in the highest temperature. DcAOX genes’ were further explored in a plant pot experiment in response to chilling, which confirmed the early AOX transcript increase prior to the induction of a specific anti-freezing gene. Our findings point to DcAOX1 and DcAOX2a as being reasonable candidates for functional marker development related to early cell reprogramming. While the genomic sequence of DcAOX2a was previously described, we characterize here the complete genomic sequence of DcAOX1.

In fungus-growing termites, fungi of the subgenus Pseudoxylaria threaten colony health through substrate competition with the termite fungus (Termitomyces). The potential mechanisms with which termites suppress Pseudoxylaria have remained unknown. Here we explore if Actinobacteria potentially play a role as defensive symbionts against Pseudoxylaria in fungus-growing termites. We sampled for Actinobacteria from 30 fungus-growing termite colonies, spanning the three main termite genera and two geographically distant sites. Our isolations yielded 360 Actinobacteria, from which we selected subsets for morphological (288 isolates, grouped in 44 morphotypes) and for 16S rRNA (35 isolates, spanning the majority of morphotypes) characterisation. Actinobacteria were found throughout all sampled nests and colony parts and, phylogenetically, they are interspersed with Actinobacteria from origins other than fungus-growing termites, indicating lack of specificity. Antibiotic-activity screening of 288 isolates against the fungal cultivar and competitor revealed that most of the Actinobacteria-produced molecules with antifungal activity. A more detailed bioassay on 53 isolates, to test the specificity of antibiotics, showed that many Actinobacteria inhibit both Pseudoxylaria and Termitomyces, and that the cultivar fungus generally is more susceptible to inhibition than the competitor. This suggests that either defensive symbionts are not present in the system or that they, if present, represent a subset of the community isolated. If so, the antibiotics must be used in a targeted fashion, being applied to specific areas by the termites. We describe the first discovery of an assembly of antibiotic-producing Actinobacteria occurring in fungus-growing termite nests. However, due to the diversity found, and the lack of both phylogenetic and bioactivity specificity, further work is necessary for a better understanding of the putative role of antibiotic-producing bacteria in the fungus-growing termite mutualistic system.

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The mutualism between fungus-growing termites (Macrotermitinae) and their mutualistic fungi (Termitomyces) began in Africa. The fungus-growing termites have secondarily colonized Madagascar and only a subset of the genera found in Africa is found on this isolated island. Successful long-distance colonization may have been severely constrained by the obligate interaction of the termites with fungal symbionts and the need to acquire these symbionts secondarily from the environment for most species (horizontal symbiont transmission). Consistent with this hypothesis, we show that all extant species of fungus-growing termites of Madagascar are the result of a single colonization event of termites belonging to one of the only two groups with vertical symbiont transmission, and we date this event at approximately 13 Mya (Middle/Upper Miocene). Vertical symbiont transmission may therefore have facilitated longdistance dispersal since both partners disperse together. In contrast to their termite hosts, the fungal symbionts have colonized Madagascar multiple times, suggesting that the presence of fungus-growing termites may have facilitated secondary colonizations of the symbiont. Our findings indicate that the absence of the right symbionts in a new environment can prevent long-distance dispersal of symbioses relying on horizontal symbiont acquisition.

We present a new perspective for the role of Termitomyces fungi in the mutualism with fungus-growing termites. According to the predominant view, this mutualism is as an example of agriculture with termites as farmers of a domesticated fungus crop, which is used for degradation of plant-material and production of fungal biomass. However, a detailed study of the literature indicates that the termites might as well be envisioned as domesticates of the fungus. According to the “ruminant hypothesis” proposed here, termite workers, by consuming asexual fruiting bodies not only harvest asexual spores, but also lignocellulolytic enzymes, which they mix with foraged plant material and enzymes of termite and possibly bacterial origin. This mixture is the building material of the fungus garden and facilitates efficient degradation of plant material. The fungus garden thus functions as an external rumen for termites and primarily the fungi themselves benefit from their own, and gut-derived, lignocellulolytic enzymes, using the termites to efficiently mix these with their growth substrate. Only secondarily the termites benefit, when they consume the degraded, nitrogen-enriched plant-fungus mixture a second time. We propose that the details of substrate use, and the degree of complementarity and redundancy among enzymes in food processing, determine selection of horizontally transmitted fungal symbionts at the start of a colony: by testing spores on a specific, mechanically and enzymatically pre-treated growth substrate, the termite host has the opportunity to select specific fungal symbionts. Potentially, the gut-microbiota thus influence host-fungus specificity, and the selection of specific fungal strains at the start of a new colony. We argue that we need to expand the current bipartite insect-biased view of the mutualism of fungus-growing termites and include the possible role of bacteria and the benefit for the fungi to fully understand the division of labor among partners in substrate degradation.

Symbiotic interactions between macrotermitine termites and their fungal symbionts have a moderate degree of specificity. Consistent with horizontal symbiont transmission, host switching has been frequent over evolutionary time so that single termite species can often be associated with several fungal symbionts. However, even in the few termite lineages that secondarily adopted vertical symbiont transmission, the fungal symbionts are not monophyletic. We addressed this paradox by studying differential transmission of fungal symbionts by alate male and female reproductives, and the genetic population structure of Termitomyces fungus gardens across 74 colonies of Macrotermes bellicosus in four west and central African countries. We confirm earlier, more limited, studies showing that the Termitomyces symbionts of M. bellicosus are normally transmitted vertically and clonally by dispersing males. We also document that the symbionts associated with this termite species belong to three main lineages that do not constitute a monophyletic group. The most common lineage occurs over the entire geographical region that we studied, including west, central and southern Africa, where it is also associated with the alternative termite hosts Macrotermes subhyalinus and Macrotermes natalensis. While Termitomyces associated with these alternative hosts are horizontally transmitted and recombine freely, the genetic population structure of the same Termitomyces associated with M. bellicosus is consistent with predominantly clonal reproduction and only occasional recombination. This implies that the genetic population structure of Termitomyces is controlled by the termite host and not by the Termitomyces symbiont.