Extreme conditions are normal for animals living in harsh environments. These animals adapt to their habitats and can use difficult conditions by default. Organisms living in enclosed spaces, notably termites in decaying wood, experience low O₂and high CO₂gas conditions due to limited gas exchange and high insect density. Termite queens, in particular, reproduce in royal chambers deep inside the wood, wherein tens of thousands of individuals engage in social labour. Here, we demonstrate that royal chambers in termite nests have low O₂and high CO₂gas concentrations, which enhance egg production by queens. We identified a unique gas condition of royal chambers in the nest of the subterranean termiteReticulitermes speratus, which is characterized by low O₂(15.75%) and high CO₂(4.99%) concentrations. Queens showed significantly greater fecundity under the low O₂and high CO₂gas conditions in the royal chambers than under ambient gas conditions. Quantitative PCR analysis revealed that the royal chamber gas conditions significantly promoted the expression levels of the vitellogenin genesRsVg1,RsVg2andRsVg3in queens compared with ambient gas conditions. This study highlights the adaptation of animals that live in closed habitats, which are hypoxic and hypercapnic as the result of their own metabolism, so as to have a high fitness in such environmental conditions.

The Clostridia is a dominant bacterial class in the guts of various animals and are considered to nutritionally contribute to the animal host. Here, we discovered clostridial endosymbionts of cellulolytic protists in termite guts, which have never been reported with evidence. We obtained (near-)complete genome sequences of three endosymbiotic Clostridia, each associated with a different parabasalid protist species with various infection rates: Trichonympha agilis, Pseudotrichonympha grassii, and Devescovina sp. All these protists are previously known to harbor permanently-associated, mutualistic Endomicrobia or Bacteroidales that supplement nitrogenous compounds. The genomes of the endosymbiotic Clostridia were small in size (1.0–1.3 Mbp) and exhibited signatures of an obligately-intracellular parasite, such as an extremely limited capability to synthesize amino acids, cofactors, and nucleotides and a disrupted glycolytic pathway with no known net ATP-generating system. Instead, the genomes encoded ATP/ADP translocase and, interestingly, regulatory proteins that are unique to eukaryotes in general and are possibly used to interfere with host cellular processes. These three genomes formed a clade with metagenome-assembled genomes (MAGs) derived from the guts of other animals, including human and ruminants, and the MAGs shared the characteristics of parasites. Gene flux analysis suggested that the acquisition of the ATP/ADP translocase gene in a common ancestor was probably key to the emergence of this parasitic clade. Taken together, we provide novel insights into the multilayered symbiotic system in the termite gut by adding the presence of parasitism and present an example of the emergence of putative energy parasites from a dominant gut bacterial clade.