Bees are well-known for their abundant biodiversity, with more than 17,500 identified species (Danforth et al., 2013). Among them, honey bees, bumblebees, and stingless bees are domesticated by humans to provide essential pollination services for crops and fruit trees (Brittain et al., 2013). These bees exhibit social behaviors and have a complete social structure, whereas most other bee species are solitary. Solitary bees have broad geographic distribution, varying body sizes, and diverse foraging preferences, making them irreplaceable pollinators in the ecological environment with flowering plants, even in places where domestic honey bee populations exist (Hedtke et al., 2013). However, the ecosystem roles of solitary bees have often been overlooked by researchers. Most studies have focused on eusocial bees, such as the honey bee Apis mellifera, while the study of solitary bees has been scarcely investigated.

The microbiota represents a vital indicator of the fitness and health of numerous insect species. Moreover, microorganisms play a critical role in many interactions between insect hosts and their habitats. For example, in the camellia weevil Curculio chinensis, the microbiota was responsible for tea saponin degradation in the insect's feeding (Zhang et al., 2020). Even though symbionts can be beneficial for their host, they can also bring negative effects to some insects. Among arthropods, Wolbachia spp. has been identified as a bacteria symbiont that distorts the reproductive cells, thereby enhancing its maternal transmission into subsequent progenies. Consequently, it has been deemed a novel pest biocontrol bacterium (Ali et al., 2018). The hispid leaf beetle Octodonta nipae is naturally infected with Wolbachia, which has been identified as an obligate endosymbiont present in all life stages, body parts, and tissues that were tested (Ali et al., 2019). Similar to other insects, the bacteria symbiont of honey bees plays a critical role in their survival. Massive losses of honey bee colonies and the decline of many bumblebee species have elicited global concern in recent decades (Lee et al., 2015; Hammer et al., 2021). A large body of evidence suggests that gut microbiota is critical in maintaining bee health (Engel et al., 2016; Jones et al., 2017; Zheng et al., 2018). Previous studies have shown that honey bees and bumblebees harbor distinctive core gut bacterial communities that are transmitted through social behaviors such as oral trophallaxis and fecal–oral pathway (Khan et al., 2020; Hammer et al., 2021). The abundance and species of core bacteria have been found to be remarkably stable, showing little effect across various habitats (Anderson et al., 2015; Kwong and Moran, 2016; Hammer et al., 2021). Host-specific bacteria have diverse functions in the digestion and absorption of nutrients, as well as in defending against pathogen colonization and reinforcing the host's immune system (Zheng et al., 2017; Kešnerová et al., 2019; Ribière et al., 2019). However, most studies have focused on interactions between gut bacteria and social bees, such as A. mellifera and Bombus terrestris, and the study of solitary bees has been scarcely investigated.

Carpenter bees belong to one taxon of wild bees (genus: Xylocopa) and are known for their large body size. They play a crucial role in crop pollination due to their greater pollination efficiency compared to honey bees in some cultivated large-flower plants such as passion fruit Passiflora edulis (Junqueira and Augusto, 2016; Alberoni et al., 2019; de Farias-Silva and Freitas, 2021). The Xylocopa genus comprises about 470 species worldwide (Michener, 2000) and 40 species mainly described in tropical and subtropical China (Wu, 2000). The social behavior of the genus Xylocopa is not well-understood. Although incipient social behaviors have been observed in some species in the wild, knowledge is lacking for most species (Handy et al., 2023). Female Xylocopa excavates their nests in dry plant tissues, such as trees, dead trunks, and bamboo canes (Junqueira et al., 2012), and lay eggs in cells with pollen and nectar (Keasar et al., 2007). Unlike social bees, nests of Xylocopa are often reused for several years (Yamamoto et al., 2014). Newly emerged female Xylocopa leave their old nest and find other abandoned nests to reproduce during the nesting seasons. Nest reusing is more common in an environment of limited nesting materials. In addition to the aforementioned characteristics, fighting between female Xylocopa for nests can also result in the reuse of old nests (He et al., 2013). Nest reusing is generally a common behavior in the genus Xylocopa.

At present, the composition of microbiota in Xylocopa species is rarely studied, with most of the microbiota species of these bees remaining unclear. However, a few available studies suggest that carpenter bees have consistent relationships with specific bacterial taxa. Xylocopa micans, Xylocopa mexicanorum, and Xylocopa tabaniformis parkinsoniae, which are carpenter bees from central Texas, were found to share similar gut bacterial communities, including Bifidobacteriaceae, Orbaceae, Lactobacillaceae, Pseudomonadaceae, and Enterobacteriaceae. Meanwhile, Xylocopa virginica had a distinct microbiota dominated by the genus Bombilactobacillus, a group of bacteria abundant in the guts of eusocial bees (Holley et al., 2022). In Xylocopa tenuiscapa, the diversity of bacteria in the foregut and hindgut were found to be different, and certain species, such as Gilliamella, Lactobacillus, and Bifidobacterium, were found to be related to those found in honey bees (Subta et al., 2020).

In the present study, we investigated the gut bacteria of two Xylocopa species (X. caerulea and X. auripennis) from different ecological environments. X. caerulea is found in Jianfengling National Forest Park of Hainan (~838 m above sea level), which is a tropical forest area rich in natural resources and one of the best protected in the region. On the other hand, X. auripennis inhabits mountainous villages with ample farmland (~650 m above see level). Symbionts are spatially organized within specific gut regions (Zheng et al., 2017, 2018). Here, we used 16S rDNA sequencing to investigate the gut bacterial communities in different parts of the intestinal tract of two Xylocopa species. Our results showed that these two carpenter bee species have consistent gut communities and specific gut symbionts that are commonly found in bumblebees. This finding provides novel insights into the symbiotic gut communities of solitary bees.

Carpenter bees are a type of plant pollinator that is covered in thick fur clumps and has a larger body size than honey bees, allowing them to carry more pollen on their bodies. They play a critical role in pollinating fruits with large flowers in tropical regions. It was previously reported that large solitary bees of the genus Xylocopa are the main pollinators for yellow passion fruit P. edulis (Barrera et al., 2020), and the supply of nests of Xylocopa frontalis in crop areas was shown to be effective for the boost of the production and quality of fruits in southeastern Brazil (Toledo et al., 2017). Beyond their commercial value in crop farming, Xylocopa bees are also recognized for their ecological importance in tropical rainforest and mangrove forests. In particular, in Malaysia's mangrove forests, Xylocopa varipuncta has been identified as a critical pollinator alongside bats and birds and plays a crucial role in carrying pollen for these ecosystems (Hodgkison et al., 2003). Generally, carpenter bees have great potential to increase fruit production and maintain the stability of the ecological system.

In the long-term evolution process, microorganisms harbor in the gut of insects with a mutually interdependent symbiotic relationship. Insects, including Apidae, rely on a mutualistic gut microbial community to digest food, detoxify harmful molecules, provide essential nutrients, protect them from pathogen and parasite invasions, and modulate development and immunity (Engel and Moran, 2013; Douglas, 2015). Eusocial bees, including honey bees (Apis) and bumblebees (Bombus), harbor host-specific and beneficial microbiota, which play multiple roles in biological activities (Kwong and Moran, 2016; Hammer et al., 2021). However, bee species vary in microbiota composition, including the presence of specialized taxa and the relative bacteria from the environment (McFrederick et al., 2017). The factors that predict this variation in microbiota composition between bee species, as well as the microbial functions that they perform, are poorly understood. However, sociality has been considered a critical driver of gut microbiota evolution in bees (Moran et al., 2019). Here we studied the gut bacterial diversity and community composition of two carpenter bees collected from a tropical rain forest, providing a more comprehensive understanding of the structure of the bacteria community in carpenter bees Xylocopa. The results reveal a complex, symbiotic community in the gut of genus Xylocopa and provide a molecular basis for understanding the function of the gut microbiota.

Specifically, we determined the bacterial composition of the top 10 most abundant phyla and families of bacteria in two carpenter bees. The results showed that the dominant gut microbiota at the phylum level in two carpenter bees was Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes, which were consistent with the previous study in honey bees (Cox-Foster et al., 2007; Khan et al., 2020). Many studies reported that Firmicutes and Proteobacteria were the foremost phyla of the microbiome in the insect gut microbiome, particularly in Hymenoptera (Jeyaprakash et al., 2003). They play a crucial role in processes, such as pectin digestion and mannose degradation, as well as in immune defense against the parasite such as Nosema bombi in bumblebees (Martinson et al., 2012; Hammer et al., 2021). The dominant families of the gut microbiome in honey bees are Lactobacillaceae, Bifidobacteriaceae, and Orbaceae. These families perform different functions, such as food digestion and nutrient absorption, which can benefit their host (Powell et al., 2014). In this study, three families dominated almost every sample we tested. As a result, our findings reveal a community composition of two carpenter bees at the phylum and family level that is similar to eusocial bees such as honey bees and bumblebees.

Eusocial bees perform vertically transmitted core gut microorganisms by the fecal–oral route, oral trophallaxis, and contact with hive materials (Powell et al., 2014). Seven bacterial species form the core microbiota of the bumblebee gut: Snodgrassella, Gilliamella, Schmidhempelia, Bifidobacterium, Bombiscardovia, Bombilactobacillus (Firm-4), and Lactobacillus (Firm-5). These species have been widely studied for their role in food digestion and nutrient absorption by their hosts (Hammer et al., 2021). In contrast, in non-social bee species, even those closely related to social corbiculates, individuals generally harbor microbiomes that are more variable and less distinct. These differences are likely due to the acquisition of microbes being driven by the environment, rather than social factors (McFrederick et al., 2012; Rothman et al., 2020). Moreover, some bee species with incomplete social structures, such as Megalopta, also possess gut core bacteria. Lactobacillus and Saccharibacter were found to be prevalent in 90% of tested adults (Graystock et al., 2017). The previous research has raised the point that environmental transmission appears to be more important than social transmission for Megalopta bees (McFrederick et al., 2014). These results reveal that some other factors, rather than social behavior, may be more critical in shaping microbiota structure and specialization. For example, Xylocopa species are considered non-classical sociality bees, and their microbiome composition has rarely been studied. However, a recent study has revealed that some incipiently social Xylocopa species also have core bacteria in their microbiomes, similar to social bees. In fact, two Xylocopa species share a set of core taxa, including Bombilactobacillus, Bombiscardovia, and Lactobacillus, which were found in most of the individual bees sampled (Handy et al., 2023). Four Xylocopa species in central Texas were found to have microbiomes dominated by bacterial taxa that were previously known only in social bees (Holley et al., 2022). In this study, several core gut bacteria of bumblebees were detected in two carpenter bees, which included Lactobacillus, Bombilactobacillus, and Bifidobacterium, and their abundance in hindguts was significantly higher than in the foregut and midgut. Lactobacillaceae, such as Lactobacillus and Bombilactobacillus, contain many genes encoding cell membrane proteins and phosphotransferase systems to assist hosts in the absorption and degradation of plant pollen (Kwong et al., 2014). In contrast, Bifidobacteriaceae degrade hexoses via a specific pathway, where the key enzymes are fructose-6-phosphate phosphoketolase (F6PPK) and xylulose phosphoketolase (Bottacini et al., 2012). The symbionts from the Lactobacillaceae and Bifidobacteriaceae were crucial for the health of honey bees, and the findings in carpenter bees were consistent with previous research (Genersch, 2010). Unexpectedly, two Bombus-specific core microbes, Candidatus Schmidhempelia and Bombiscardovia, were abundant in the hindguts of two carpenter bees. Candidatus Schmidhempelia is a Bombus-specific gamma-proteobacteria, which has been found in 90% of bumblebee guts and has a simplified genome unique to symbiotic bacteria (Martinson et al., 2014). Bombiscardovia is a genus of Bifidobacteriaceae originally isolated from Bombus lapidaries (Killer et al., 2010). Although the functions of the two genera have not been studied extensively, they are considered beneficial to their hosts. Our results, when compared to the reported gut microbiota of carpenter bees and eusocial bees, show that the genus Lactobacillus and Bifidobacterium are commonly found in the gut of all studied genus Xylocopa. In contrast, the genus Bombilactobacillus, Gilliamella, Bombiscardovia, and Schmidhempelia are inconsistently distributed in the guts of carpenter bees, and the genus Snodgrassella is only found in the guts of eusocial bees. In addition to the core bacteria, the genera Leuconostoc and Apilactobacillus were found in significantly higher abundance compared to other bacteria. The bacterial taxon known as Apilactobacillus was found to be common in the gut and provisions of solitary bees, as well as in the crop of bees (McFrederick et al., 2017). Hypotheses suggest that Apilactobacillus may have the ability to inhibit the growth of pathogens or prevent the spoilage of stored pollen (McFrederick et al., 2018).

The specific composition and spatial distribution of the gut microbiota in eusocial bees depend on their functional differentiation (Jeyaprakash et al., 2003). In this study, the differences in bacteria species between the fore-midguts and hindguts of the two carpenter bees were tested by LEfSe analysis. The members of Lactobacillaceae and Bifidobacteriaceae, such as the genera Bombilactobacillus, Lactobacillus, and Bifidobacterium, were significantly enriched in hindguts. This finding is consistent with the results observed in social bees. In honey bees A. mellifera, the hindguts function in fecal storage, reabsorption of nutrients, and enabling the colonization of core bacteria, such as Bombilactobacillus, Lactobacillus, and Bifidobacterium, which have enriched genes participating in the carbohydrate uptake and metabolism pathways (Kwong and Moran, 2016). The predicted functional pathways, tested by PICRUSt2, in the hindguts of X. caerulea and X. auripennis were concentrated in carbohydrate and lipid metabolic processes, which corresponded with bacterial species distributed in the hindguts. These results suggest that Lactobacillaceae and Bifidobacteriaceae are dominant in the hindguts and may play a vital role in carbohydrate and lipid metabolism. PICRUSt2 contains an updated and large database of gene families and reference genomes and provides interoperability with any operational taxonomic unit (OTU)-picking or denoising algorithm (Douglas et al., 2020). A large number of studies have used this method to predict the functions of gut microbiota. For example, in the oriental fruit moth Grapholita molesta, feeding on different plants can significantly change the functions of gut microbiota (Yuan et al., 2021). However, there are still some limitations when using PICRUSt2 to predict based on a fragment of the V3–V4 region. Future experiments should be designed to use metagenomic or single bacterial genome approaches to conduct more intensive studies.

In eusocial bees, such as honey bees, the core microbiota can be vertically transmitted between workers and larvae through trophallaxis and the fecal–oral pathway (Powell et al., 2014). In contrast, the gut microbes of solitary bees are believed to be acquired from the hive environment and food due to the absence of close social contact (Gilliam et al., 1984). There is mounting evidence that some bacteria taxa, previously known only in social bees, exist in the guts of solitary bees and may have recently transmitted from mother bees to larvae. Previous studies have shown that Bifidobacterium isolates from the guts of European X. violacea were closely related to those of honey bees and bumblebees (Alberoni et al., 2019). In this study, we found that X. caerulea and X. auripennis shared similar bee-associated bacterial community profiles despite inhabiting different ecological environments. Lactobacillaceae, Orbaceae, and Bifidobacteriaceae were the three main families of gut microbiota in bees, including the genera Lactobacillus, Apilactobacillus, Bombilactobacillus, Candidatus Schmidhempelia, Bifidobacterium, and Bombiscardovia. Most of the ASVs found in carpenter bees of the three families in this study were closely related to previously identified bacterial taxa, which are widespread in social bees, particularly Bombus-specific genera Candidatus Schmidhempelia and Bombiscardovia. These results suggest that the vertical transmission of bacteria in Xylocopa may occur through certain mechanisms.

It is commonly believed that solitary bees exhibit no caring behavior, with mother bees only collecting food for the larvae and leaving the hive before the offspring mature. The gut microorganisms of their offspring are mainly acquired from the environment and from food sources, such as the genera Megachile and Osmia (Keller et al., 2018; Voulgari-Kokota et al., 2019). However, a previous study of the bee species in the genera Megalopta (which contains solitary and social species) found a limited influence of sociality on bacterial composition (McFrederick et al., 2014). This demonstrated that microbiota was not only transmitted by direct social contacts, such as trophallaxis and fecal–oral contact, between concurrent members but also by some non-social behaviors. Furthermore, the vertical transmission of core gut bacteria in two carpenter bee species may be linked to other observed behaviors within the Xylocopa genus. In X. sulcatipes, different generations of mother bees fight for nest chambers due to competition in a resource-limited environment (Stark, 2010). The usurper drives the host out of the hive or defense against the enemy instead of reproducing and laying new eggs in the used hive, which probably drives microbe–host specificity by contact with old hive materials. In the social bee A. mellifera, newly emerged young honey bees chew their way out of cells and consume gut core microbiota that remained on hive surfaces (Martinson et al., 2012). Recent research has confirmed that the transmission of honey bee core hindgut microbiome is facultative and horizontal, with five out of six core hindgut species readily acquired from the built hive structure and natural diet (Anderson et al., 2022). The same route of transmission may exist in the genus Xylocopa and result in an accumulation of bacterial species in the guts of young bees from old hive materials. In this study, we collected samples of two carpenter bees in a relatively high population density region where nests are concentrated. Previous studies have predominantly collected solitary bees randomly in the wild, where there is minimal competition pressure and unstable gut microbiota. Thus, our findings indicate that the pressures of nesting and reproduction for Xylocopa seem to drive the reuse of old nests and the vertical transmission of gut bacteria, although the life habits of most Xylocopa species are poorly studied. The social transmission routes of Xylocopa species merit further investigation.

In conclusion, we characterized the gut microbial communities of two carpenter bees and found that some gut bacterial taxa exist in the guts of X. caerulea and X. auripennis, such as Candidatus Schmidhempelia and Bombiscardovia, which were closely related to those found in eusocial bees, especially bumblebees. Based on our results, we hypothesize that the gut bacteria of carpenter bees are transmitted from mother bees to larvae by reusing old nests. This study offers novel insight into the structure, distribution, and function of gut symbiotic bacteria in Xylocopa species. However, there were still some limitations in our study. Future experiments should be designed to compare the gut microbiota of these two carpenter bees with that of other species in the genus Xylocopa and eusocial bees. Moreover, isolating Candidatus Schmidhempelia and Bombiscardovia from the two Xylocopa species and elucidating their important functions using multi-omics will contribute to finding new probiotics that people can use.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: NCBI—PRJNA915489.

WH and YG designed the research and wrote the manuscript. YG, YW, SZ, and DL collected the samples. WH, YG, SW, and YZ performed the experiments and analyzed the data. WH and JG funded this study. All authors contributed to the article and approved the submitted version.