Animals harbor gut microorganisms that have evolved along with host lineages, including humans, chimpanzees, gorillas, and orangutans. This provides evidence for the co-diversification of hosts and some lineages of the gut bacteria, implying a long-term vertical association (Moeller et al., 2016; Moran et al., 2019). Similarly, in social corbiculate bees (honey bees, bumble bees, and stingless bees), five core lineages of the gut microbiota show phylogenies mostly matching their hosts, supporting co-diversification over approximately 80 million years (Kwong et al., 2017). The five core gut bacteria, Gilliamella, Snodgrassella, Bifidobacterium, Lactobacillus Firm-4, and Lactobacillus Firm-5, have undergone functional evolution to adapt to the host. By utilizing their own metabolic capabilities, the gut microbiota help hosts expand their ecological range, which benefits them (Kwong and Moran, 2016). A previous study has indicated that symbiotic bacteria in insects are essential for host health by contributing to food digestion, detoxifying toxic molecules, providing essential nutrients, and protecting against pathogens and parasites (Engel and Moran, 2013; Zheng et al., 2016; Mockler et al., 2018; Nishida and Ochman, 2018).

Bumblebees are important pollinators of wild plants and crops. China has ~50% of the world's species of bumblebees and is the country with the richest bumblebee species (Huang and An, 2018). China is known for its abundant and distinct species of bumblebees, such as Bombus pyrosoma, which is found across a wide range, stretching from western Liaoning to eastern Qinghai provinces, and has also been reported in provinces such as Shanxi, Hebei, and Gansu (An et al., 2008; Wu et al., 2009). Their habitats span different ecosystems, from low elevations in the North China Plain to high elevations in the Qinghai–Tibet Plateau (An et al., 2014; Williams et al., 2016). Bumblebees prefer cooler climates, leading to greater diversity in the temperate habitats of the Northern Hemisphere, particularly across Eurasia. In contrast, bumblebee species inhabiting tropical regions tend to be restricted to higher elevations due to the more favorable conditions found there (Engel and Rasmussen, 2020).

High-altitude areas such as the Qinghai-Tibetan Plateau are often accompanied by extreme weather conditions of low temperature, hypoxia, and strong ultraviolet radiation, which are usually unsuitable for the growth and development of biological organisms (Yu et al., 2023). However, organisms such as mammals and ruminants have evolved some strategies to adapt to high-altitude environments (Friedrich and Wiener, 2020). One strategy for dealing with low oxygen levels is to increase oxygen availability in mammals (Storz et al., 2010). For example, populations at high altitudes have lower ventilation than populations at low altitudes (Brutsaert, 2007). In addition, oxygen affinity is enhanced by increasing red blood cell count and hemoglobin level (Weber, 2007). Another strategy is to reduce the body's need for oxygen by lowering its overall metabolic rate (Hochachka et al., 1996). Studies have shown that the energy expenditure of native Andean llamas is significantly lower compared to other ruminants (Riek et al., 2019). High-altitude areas not only affect mammals but also their symbiotic microbiota. Compared to low-altitude individuals, high-altitude species such as humans, Tibetan sheep, and pigs have different compositions and diversity of their symbiotic microbiota (Yang et al., 2017; Ma et al., 2019). However, the metabolic role of the gut microbiota in animals living in high-altitude environments and the potential co-evolution between gut microbiota and host in high-altitude adaptation have not yet been fully understood.

The interaction between gut microbiota not only regulates the organism's health but also forms a crucial bridge between the environment and the host, helping the host to better adapt to the environment (Gao et al., 2020). The unique habitat conditions and simple gut patterns of Bombus pyrosoma provide an excellent opportunity to study how gut microbiota help the host adapt to high-altitude evolutionary pressures (Zhang and Zheng, 2022). We hypothesize that the composition and abundance of gut microbiota from Bombus pyrosoma in high-altitude areas may differ and help the host improve its ability to adapt to high-altitude environments.

Compared with bumblebees in low-altitude regions, the gut bacteria of B. pyrosoma in high-altitude areas have lower alpha diversity and a relatively uniform community composition, which may result from collecting only a single type of plant pollen and nectar. Apart from host attributes (e.g., species, age, and caste) determining the gut microbiota of bumblebees, selective forces (e.g., antibiotic-resistant genes and habitat ecological environment) may also play an important role in shaping the formation of the gut microbial community (Colman et al., 2012; Kwong et al., 2014; Kwong and Moran, 2015, 2016; Zhang et al., 2021). For example, differences in external environments lead to different microbial community structures in wild and artificially reared bee colonies, with wild colonies having more non-core microbial groups (Zhang and Zheng, 2022). Owing to the obvious difference in eco-climate between high- and low-altitude areas, low temperature, hypoxia, ultraviolet radiation, and other climatic characteristics in low-altitude areas lead to the decline of plant diversity (Bruns and Kennedy, 2009; Murray, 2016). However, plant diversity is higher in low-altitude areas, where the gut microbiota displays higher diversity and has a more dispersed community composition.

The high-altitude regions have fewer Fructobacillus and Saccharibacter. It may be because there are fewer carbon sources at higher altitudes. The gut microbiota, such as Fructobacillus and Saccharibacter, generally benefit the health of their hosts. Members of the genus Frugibacillus are classified as frugiophilic lactic acid bacteria (Endo et al., 2018). The fructose content found in the nectar of quince cultivars from undamaged varieties was higher than that from cultivars found under frozen conditions (Akšić et al., 2014). To adapt to fructose-rich environments such as fruit and honey-rich flowers, Fructobacillus underwent a specific reductive evolution similar to that of Lactobacillus kunkeei (Lee et al., 2015; Maeno et al., 2016). Frutobacillus and Saccharibacter, present in the bumblebee gut, help the host degrade fructose through carbohydrate catabolism (Praet et al., 2018). The genus Saccharibacter is an acetic acid bacterium that has been shown to be associated with a variety of insects that rely on a high-sugar diet (Crotti et al., 2010; Chouaia et al., 2014). Since the genus Saccharibacter is isolated from flowers, it may also come into contact with insect pollinators and play a role in their use of nectar as a food source (Smith et al., 2020). However, some individuals in low-altitude areas even have a predominance of opportunistic bacteria such as Serratia, which may indicate that bumblebees in low-altitude areas face more severe environments such as ecological pollution.

Plateau-adapted hosts have developed strategies to cope with adverse environments, such as lowering their overall metabolic rate to reduce the body's demand for oxygen (Moore, 2017). Under low temperatures and hypoxic conditions, insects upregulate the expression of immune-related genes, such as those responsible for encoding antimicrobial peptides and the activation of the IKK/NF-kB signaling pathway, which in turn stimulates the immune response (Zhang et al., 2011; Xu and James, 2012; Bandarra et al., 2014). Compared to gut bacteria of bumblebee individuals in low-altitude areas, those in high-altitude regions have a higher abundance of pathways associated with “One carbon pool by folate,” “Antifolate resistance,” “Protein digestion and absorption,” and “Biosynthesis of amino acids.” Conversely, there is a decrease in pathways related to “Carbohydrate digestion and absorption” and “Salmonella infection.” This variation may be due to adverse environmental conditions such as low temperatures, hypoxia, and pollen and nectar food shortages at high altitudes. The Tibetan B. pyrosoma reduces energy metabolism and enhances immunity, which necessitates fewer carbohydrates and more protein to cope with these challenges. Correspondingly, the gut microbiota regulates members' species and abundance (e.g., Fructobacillus and Saccharibacter), exerting less demand for carbohydrates and improving protein utilization to help the host cope with an adverse high-altitude environment.

The gut microbiota co-evolved with their hosts for over 80 million years. Intestinal bacteria expand the host's ecological range through their metabolic capacities. Similarly, the host's ecological environment can also affect the composition and abundance of intestinal bacteria. Hosts at high altitudes, such as Bombus pyrosoma, adapt to the plateau environment by reducing energy metabolism and improving immunity. In turn, the gut microbiota helps the host adapt to the adverse environment by regulating their members' abundance and functional metabolism, such as reducing carbohydrate degradation and improving the utilization of protein. Our findings provide insights into the interactions between the bumblebee gut microbiota and the host.

The PacBio circular consensus sequencing (CCS) reads have been deposited in the Genome Sequence Archive in the BIG Data Center, Beijing Institute of Genomics (BIG), Chinese Academy of Sciences, under accession numbers CRA010937 that are publicly accessible at http://bigd.big.ac.cn/gsa, accessed on 7 May 2023.

ZheZ and FY conceived the research project. ZheZ, MZ, FL, ZX, ZhiZ, HZ, YW, and JH collected the samples, analyzed the data, and sequenced the samples. ZheZ, YG, and JL wrote the manuscript. All authors read and approved the final version of the manuscript.