Heat and desiccation tolerances predict bee abundance under climate change.

Climate change could pose an urgent threat to pollinators, with critical ecological and economic consequences. However, for most insect pollinator species, we lack the long-term data and mechanistic evidence that are necessary to identify climate-driven declines and predict future trends. Here we document 16 years of abundance patterns for a hyper-diverse bee assemblage1 in a warming and drying region2, link bee declines with experimentally determined heat and desiccation tolerances, and use climate sensitivity models to project bee communities into the future. Aridity strongly predicted bee abundance for 71% of 665 bee populations (species × ecosystem combinations). Bee taxa that best tolerated heat and desiccation increased the most over time. Models forecasted declines for 46% of species and predicted more homogeneous communities dominated by drought-tolerant taxa, even while total bee abundance may remain unchanged. Such community reordering could reduce pollination services, because diverse bee assemblages typically maximize pollination for plant communities3. Larger-bodied bees also dominated under intermediate to high aridity, identifying body size as a valuable trait for understanding how climate-driven shifts in bee communities influence pollination4. We provide evidence that climate change directly threatens bee diversity, indicating that bee conservation efforts should account for the stress of aridity on bee physiology.

Body-size-dependent effects of landscape-level resource energetics on pollinator abundance in woodland remnants.

Land use change alters floral resource availability, thereby contributing to declines in important pollinators. However, the severity of land use impact varies by species, influenced by factors such as dispersal ability and resource specialization, both of which can correlate with body size. Here. we test whether floral resource availability in the surrounding landscape (the 'matrix') influences bee species' abundance in isolated remnant woodlands, and whether this effect varies with body size. We sampled quantitative flower-visitation networks within woodland remnants and quantified floral energy resources (nectar and pollen calories) available to each bee species both within the woodland and the matrix. Bee abundance in woodland increased with floral energy resources in the surrounding matrix, with strongest effects on larger-bodied species. Our findings suggest important but size-dependent effects of declining matrix floral resources on the persistence of bees in remnant woodlands, highlighting the need to incorporate landscape-level floral resources in conservation planning for pollinators in threatened natural habitats.

Essential role of papillae flexibility in nectar capture by bees.

Many bees possess a tongue resembling a brush composed of a central rod (glossa) covered by elongated papillae, which is dipped periodically into nectar to collect this primary source of energy. In vivo measurements show that the amount of nectar collected per lap remains essentially constant for sugar concentrations lower than 50% but drops significantly for a concentration around 70%. To understand this variation of the ingestion rate with the sugar content of nectar, we investigate the dynamics of fluid capture by Bombus terrestris as a model system. During the dipping process, the papillae, which initially adhere to the glossa, unfold when immersed in the nectar. Combining in vivo investigations, macroscopic experiments with flexible rods, and an elastoviscous theoretical model, we show that the capture mechanism is governed by the relaxation dynamics of the bent papillae, driven by their elastic recoil slowed down through viscous dissipation. At low sugar concentrations, the papillae completely open before the tongue retracts out of nectar and thus, fully contribute to the fluid capture. In contrast, at larger concentrations corresponding to the drop of the ingestion rate, the viscous dissipation strongly hinders the papillae opening, reducing considerably the amount of nectar captured. This study shows the crucial role of flexible papillae, whose aspect ratio determines the optimal nectar concentration, to understand quantitatively the capture of nectar by bees and how physics can shed some light on the degree of adaptation of a specific morphological trait.

Anatomical, phenological and genetic aspects of the host-parasite relationship between Andrena vaga (Hymenoptera) and Stylops ater (Strepsiptera).

Stylops ater is an endoparasite of the mining bee Andrena vaga with extreme sexual dimorphism and hypermetamorphosis. Its population structure, parasitization mode, genetic diversity and impact on host morphology were examined in nesting sites in Germany to better understand this highly specialized host­parasite interaction. The shift in host emergence due to stylopization was proven to be especially strong in A. vaga. Around 10% of bees hosted more than 1 Stylops, with at maximum 4. A trend in Stylops' preference for hosts of their own sex and a sex-specific position of extrusion from the host abdomen was found. Invasion of Andrena eggs by Stylops primary larvae was depicted for the first time. Cephalothoraces of female Stylops were smaller in male and pluristylopized hosts, likely due to lower nutrient supply. The genes H3, 18S and cytochrome c oxidase subunit 1 were highly conserved, revealing near-absence of local variation within Stylops. Ovaries of hosts with male Stylops contained poorly developed eggs while those of hosts with female Stylops were devoid of visible eggs, which might be due to a higher protein demand of female Stylops. Male Stylops, which might have a more energy-consuming development, led to a reduction in head width of their hosts. Host masculinization was present in the leaner shape of the metabasitarsus of stylopized females and is interpreted as a by-product of manipulation of the host's endocrine system to shift its emergence. Stylopization intensified tergal hairiness, most strongly in hosts with female Stylops, near the point of parasite extrusion, hinting towards substance-induced host manipulation.

Bumblebees perceive the spatial layout of their environment in relation to their body size and form to minimize inflight collisions.

Animals that move through complex habitats must frequently contend with obstacles in their path. Humans and other highly cognitive vertebrates avoid collisions by perceiving the relationship between the layout of their surroundings and the properties of their own body profile and action capacity. It is unknown whether insects, which have much smaller brains, possess such abilities. We used bumblebees, which vary widely in body size and regularly forage in dense vegetation, to investigate whether flying insects consider their own size when interacting with their surroundings. Bumblebees trained to fly in a tunnel were sporadically presented with an obstructing wall containing a gap that varied in width. Bees successfully flew through narrow gaps, even those that were much smaller than their wingspans, by first performing lateral scanning (side-to-side flights) to visually assess the aperture. Bees then reoriented their in-flight posture (i.e., yaw or heading angle) while passing through, minimizing their projected frontal width and mitigating collisions; in extreme cases, bees flew entirely sideways through the gap. Both the time that bees spent scanning during their approach and the extent to which they reoriented themselves to pass through the gap were determined not by the absolute size of the gap, but by the size of the gap relative to each bee's own wingspan. Our findings suggest that, similar to humans and other vertebrates, flying bumblebees perceive the affordance of their surroundings relative their body size and form to navigate safely through complex environments.

The Neuroproteomic Basis of Enhanced Perception and Processing of Brood Signals That Trigger Increased Reproductive Investment in Honeybee (Apis mellifera) Workers.

The neuronal basis of complex social behavior is still poorly understood. In honeybees, reproductive investment decisions are made at the colony-level. Queens develop from female-destined larvae that receive alloparental care from nurse bees in the form of ad-libitum royal jelly (RJ) secretions. Typically, the number of raised new queens is limited but genetic breeding of "royal jelly bees" (RJBs) for enhanced RJ production over decades has led to a dramatic increase of reproductive investment in queens. Here, we compare RJBs to unselected Italian bees (ITBs) to investigate how their cognitive processing of larval signals in the mushroom bodies (MBs) and antennal lobes (ALs) may contribute to their behavioral differences. A cross-fostering experiment confirms that the RJB syndrome is mainly due to a shift in nurse bee alloparental care behavior. Using olfactory conditioning of the proboscis extension reflex, we show that the RJB nurses spontaneously respond more often to larval odors compared with ITB nurses but their subsequent learning occurs at similar rates. These phenotypic findings are corroborated by our demonstration that the proteome of the brain, particularly of the ALs differs between RJBs and ITBs. Notably, in the ALs of RJB newly emerged bees and nurses compared with ITBs, processes of energy and nutrient metabolism, signal transduction are up-regulated, priming the ALs for receiving and processing the brood signals from the antennae. Moreover, highly abundant major royal jelly proteins and hexamerins in RJBs compared with ITBs during early life when the nervous system still develops suggest crucial new neurobiological roles for these well-characterized proteins. Altogether, our findings reveal that RJBs have evolved a strong olfactory response to larvae, enabled by numerous neurophysiological adaptations that increase the nurse bees' alloparental care behavior.

Neuroanatomical differentiation associated with alternative reproductive tactics in male arid land bees, Centris pallida and Amegilla dawsoni.

Alternative reproductive tactics (ARTs) occur when there is categorical variation in the reproductive strategies of a sex within a population. These different behavioral phenotypes can expose animals to distinct cognitive challenges, which may be addressed through neuroanatomical differentiation. The dramatic phenotypic plasticity underlying ARTs provides a powerful opportunity to study how intraspecific nervous system variation can support distinct cognitive abilities. We hypothesized that conspecific animals pursuing ARTs would exhibit dissimilar brain architecture. Dimorphic males of the bee species Centris pallida and Amegilla dawsoni use alternative mate location strategies that rely primarily on either olfaction (large-morph) or vision (small-morph) to find females. This variation in behavior led us to predict increased volumes of the brain regions supporting their primarily chemosensory or visual mate location strategies. Large-morph males relying mainly on olfaction had relatively larger antennal lobes and relatively smaller optic lobes than small-morph males relying primarily on visual cues. In both species, as relative volumes of the optic lobe increased, the relative volume of the antennal lobe decreased. In addition, A. dawsoni large males had relatively larger mushroom body lips, which process olfactory inputs. Our results suggest that the divergent behavioral strategies in ART systems can be associated with neuroanatomical differentiation.

Body and Wing Allometries Reveal Flight-Fecundity Tradeoff in Response to Larval Provisioning in Osmia lignaria (Hymenoptera: Megachilidae).

Variation in body size has important implications for physical performance and fitness. For insects, adult size and morphology are determined by larval growth and metamorphosis. Female blue orchard bees, Osmia lignaria, (Say) provision a finite quantity of food to their offspring. In this study, we asked how provision-dependent variation in size changes adult morphology. We performed a diet manipulation in which some larvae were starved in the final instar and some were given unlimited food. We examined the consequences on adult morphology in two ways. First, allometric relationships between major body regions (head, thorax, abdomen) and total body mass were measured to determine relative growth of these structures. Second, morphometrics that are critical for flight (wing area, wing loading, and extra flight power index) were quantified. Head and thorax mass had hyperallometric relationships with body size, indicating these parts become disproportionately large in adults when larvae are given copious provisions. However, abdominal mass and wing area increased hypoallometrically with body size. Thus, large adults had disproportionately lighter abdomens and smaller wing areas than smaller adults. Though both males and females followed these general patterns, allometric patterns were affected by sex. For flight metrics, small adults had reduced wing loading and an increased extra flight power index. These results suggest that diet quantity alters development in ways that affect the morphometric trait relationships in adult O. lignaria and may lead to functional differences in performance.

Genetics of reproduction and regulation of honeybee (Apis mellifera L.) social behavior.

Honeybees form complex societies with a division of labor for reproduction, nutrition, nest construction and maintenance, and defense. How does it evolve? Tasks performed by worker honeybees are distributed in time and space. There is no central control over behavior and there is no central genome on which selection can act and effect adaptive change. For 22 years, we have been addressing these questions by selecting on a single social trait associated with nutrition: the amount of surplus pollen (a source of protein) that is stored in the combs of the nest. Forty-two generations of selection have revealed changes at biological levels extending from the society down to the level of the gene. We show how we constructed this vertical understanding of social evolution using behavioral and anatomical analyses, physiology, genetic mapping, and gene knockdowns. We map out the phenotypic and genetic architectures of food storage and foraging behavior and show how they are linked through broad epistasis and pleiotropy affecting a reproductive regulatory network that influences foraging behavior. This is remarkable because worker honeybees have reduced reproductive organs and are normally sterile; however, the reproductive regulatory network has been co-opted for behavioral division of labor.

Characterization of the olfactory system of the giant honey bee, Apis dorsata.

Apis dorsata is an open-nesting, undomesticated, giant honey bee found in southern Asia. We characterized a number of aspects of olfactory system of Apis dorsata and compared it with the well-characterized, western honeybee, Apis mellifera, a domesticated, cavity-nesting species. A. dorsata differs from A. mellifera in nesting behavior, foraging activity, and defense mechanisms. Hence, there can be different demands on its olfactory system. We elucidated the glomerular organization of A. dorsata by creating a digital atlas for the antennal lobe and visualized the antennal lobe tracts and localized their innervations. We showed that the neurites of Kenyon cells with cell bodies located in a neighborhood in calyx retain their relative neighborhoods in the pedunculus and the vertical lobe forming a columnar organization in the mushroom body. The vertical lobe and the calyx of the mushroom body were found to be innervated by extrinsic neurons with cell bodies in the lateral protocerebrum. We found that the species was amenable to olfactory conditioning and showed good learning and memory retention at 24 h after training. It was also amenable to massed and spaced conditioning and could distinguish trained odor from an untrained novel odor. We found that all the above mentioned features in A. dorsata are very similar to those in A. mellifera. We thereby establish A. dorsata as a good model system, strikingly similar to A. mellifera despite the differences in their nesting and foraging behavior.

Temporal model of fluid-feeding mechanisms in a long proboscid orchid bee compared to the short proboscid honey bee.

Bees (Apidae) are flower-visiting insects that possess highly efficient mouthparts for the ingestion of nectar and other sucrose fluids. Their mouthparts are composed of mandibles and a tube-like proboscis. The proboscis forms a food canal, which encompasses a protrusible and hairy tongue to load and imbibe nectar, representing a fluid-feeding technique with a low Reynolds number. The western honey bee, Apis mellifera ligustica, can rhythmically erect the tongue microtrichia to regulate the glossal shape, achieving a tradeoff between nectar intake rate and viscous drag. Neotropical orchid bees (Euglossa imperialis) possess a proboscis longer than the body and combines this lapping-sucking mode of fluid-feeding with suction feeding. This additional technique of nectar uptake may have different biophysics. In order to reveal the effect of special structures of mouthparts in terms of feeding efficiency, we build a temporal model for orchid bees considering fluid transport in multi-states including active suction, tongue protraction and viscous dipping. Our model indicates that the dipping technique employed by honey bees can contribute to more than seven times the volumetric and energetic intake rate at a certain nectar concentration compared with the combined mode used by orchid bees. The high capability of the honey bee's proboscis to ingest nectar may inspire micropumps for transporting viscous liquid with higher efficiency.

Intraspecific Variation in Worker Body Size Makes North American Bumble Bees (Bombus spp.) Less Susceptible to Decline.

Population declines have been documented in approximately one-third of bumble bee species. Certain drivers of these declines are known; however, less is known about the interspecific trait differences that make certain species more susceptible to decline. Two traits that have implications for responding to rapidly changed environments may be particularly consequential for bumble bee populations: intraspecific body size variation and brain size. Bumble bee body size is highly variable and is likely adaptive at the colony level, and brain size correlates with cognitive traits (e.g., behavioral plasticity) in many groups. Trait variation and plasticity may buffer species against negative effects of rapidly changed environments. Using phylogenetically controlled analyses of 31 North American bumble bee species, we find that higher intraspecific body size variation is associated with species having increased their relative abundance over time. However, this variation does not significantly interact with tongue length, another trait thought to influence bees' decline susceptibility. Head size, a proxy for brain size, is not correlated with change in relative abundance. Our results support the hypothesis that variation in body size makes species less susceptible to decline in rapidly altered environments and suggests that this variation is important to the success of bumble bee populations.

In-hive variation of the gut microbial composition of honey bee larvae and pupae from the same oviposition time.

BACKGROUND: Knowledge of microbiota composition, persistence, and transmission as well as the overall function of the bacterial community is important and may be linked to honey bee health. This study aimed to investigate the inter-individual variation in the gut microbiota in honey bee larvae and pupae. RESULTS: Individual larvae differed in the composition of major bacterial groups. In the majority of 5th instar bees, Firmicutes showed predominance (70%); however, after larval defecation and during pupation, the abundance decreased to 40%, in favour of Gammaproteobacteria. The 5th instar larvae hosted significantly more (P < 0.001) Firmicutes than black pupae. Power calculations revealed that 11 and 18 replicate-individuals, respectively, were required for the detection of significant differences (P < 0.05) in the Bacteroidetes and Firmicutes abundance between stages, while higher numbers of replicates were required for Actinobacteria (478 replicates) and Gammaproteobacteria (111 replicates). CONCLUSIONS: Although sample processing and extraction protocols may have had a significant influence, sampling is very important for studying the bee microbiome, and the importance of the number of individuals pooled in samples used for microbiome studies should not be underestimated.

Brain differences between social castes precede group formation in a primitively eusocial bee.

Social interactions may shape brain development. In primitively eusocial insects, the mushroom body (MB), an area of the brain associated with sensory integration and learning, is larger in queens than in workers. This may reflect a strategy of neural investment in queens or it may be a plastic response to social interactions in the nest. Here, we show that nest foundresses-the reproductive females who will become queens but are solitary until their first workers are born-have larger MBs than workers in the primitively eusocial sweat bee Augochlorella aurata. Whole brain size and optic lobe size do not differ between the two groups, but foundresses also have larger antennal lobes than workers. This shows that increased neural investment in MBs precedes social group formation. Larger MBs among foundresses may reflect the increased larval nutrition provisioned to future queens and the lack of social aggression from a dominant queen upon adult emergence.

Body mass but not wing size or symmetry correlates with life span of honey bee drones.

In social insects such as the honey bee, the quality of drones at the time of their emergence can affect their maintenance in the colony until maturity. Body mass, wing size and wing asymmetry of emerging honey bee drones were measured and correlated with their life span in the colony and compared between individuals reaching maturity or not. The life span of drones differed among colonies in which they were maintained after emergence but not between colonies in which they were reared. More drones heavier at emergence reached sexual maturity at 15 days and had a longer life span compared with light-weight drones of lower mass. The size and symmetry of drone forewings was not correlated with their life span. Our results suggest that body mass at emergence is a good predictor of drone survival in the colony.

Optimized Histological Preparation of Ovary for Ovariole Counting in Africanized Honey Bee Queens (Hymenoptera: Apidae).

Techniques for counting ovariole number in virgin and mated Apis mellifera L. queens have been described in previous studies. Having a systematic and fast way to collect this measurement can help accelerate bee breeding programs, because selection decisions can be taken faster. The aim of this work was to develop an efficient histological method to preserve ovaries that allows assessing the number of ovarioles in newly emerged virgin queens, and also in mated queens, in a shorter time than the methods already published. The proposed method resulted in images suitable for ovariole counting in both newly emerged and mated queens, and the total histological process took less than 10 h. This method provides the optimization of the histological procedure for research breeding programs that use ovariole number as selection criteria for improving reproduction and production traits.

Post-embryonic changes in the hindgut of honeybee Apis mellifera workers: Morphology, cuticle deposition, apoptosis, and cell proliferation.

In insects, the hindgut is a homeostatic region of the digestive tract, divided into pylorus, ileum, and rectum, that reabsorbs water, ions, and small molecules produced during hemolymph filtration. The hindgut anatomy in bee larvae is different from that of adult workers. This study reports the morphological changes and cellular events that occur in the hindgut during the metamorphosis of the honeybee Apis mellifera. We describe the occurrence of autophagosomes and the ultrastructure of the epithelial cells and cuticle, suggesting that cuticular degradation begins in prepupae, with the cuticle being reabsorbed and recycled by autophagosomes in white- and pink-eyed pupae, followed by the deposition of new cuticle in light-brown-eyed pupae. In L5S larvae and prepupae, the hindgut undergoes cell proliferation in the anterior and posterior ends. In the pupae, the pylorus, ileum, and rectum regions are differentiated, and cell proliferation ceases in dark-brown-eyed pupae. Apoptosis occurs in the hindgut from the L5S larval to the pink-eyed pupal stage. In light-brown- and dark-brown-eyed pupae, the ileum epithelium changes from pseudostratified to simple only after the production of the basal lamina, whereas the rectal epithelium is always flattened. In black-eyed pupae, ileum epithelial cells have large vacuoles and subcuticular spaces, while in adult forager workers these cells have long invaginations in the cell apex and many mitochondria, indicating a role in the transport of compounds. Our findings show that hindgut morphogenesis is a dynamic process, with tissue remodeling and cellular events taking place for the formation of different regions of the organ, the reconstruction of a new cuticle, and the remodeling of visceral muscles.

Population genomics and morphometric assignment of western honey bees (Apis mellifera L.) in the Republic of South Africa.

BACKGROUNDS: Apis mellifera scutellata and A.m. capensis (the Cape honey bee) are western honey bee subspecies indigenous to the Republic of South Africa (RSA). Both bees are important for biological and economic reasons. First, A.m. scutellata is the invasive "African honey bee" of the Americas and exhibits a number of traits that beekeepers consider undesirable. They swarm excessively, are prone to absconding (vacating the nest entirely), usurp other honey bee colonies, and exhibit heightened defensiveness. Second, Cape honey bees are socially parasitic bees; the workers can reproduce thelytokously. Both bees are indistinguishable visually. Therefore, we employed Genotyping-by-Sequencing (GBS), wing geometry and standard morphometric approaches to assess the genetic diversity and population structure of these bees to search for diagnostic markers that can be employed to distinguish between the two subspecies. RESULTS: Apis mellifera scutellata possessed the highest mean number of polymorphic SNPs (among 2449 informative SNPs) with minor allele frequencies > 0.05 (Np = 88%). The RSA honey bees generated a high level of expected heterozygosity (Hexp = 0.24). The mean genetic differentiation (FST; 6.5%) among the RSA honey bees revealed that approximately 93% of the genetic variation was accounted for within individuals of these subspecies. Two genetically distinct clusters (K = 2) corresponding to both subspecies were detected by Model-based Bayesian clustering and supported by Principal Coordinates Analysis (PCoA) inferences. Selected highly divergent loci (n = 83) further reinforced a distinctive clustering of two subspecies across geographical origins, accounting for approximately 83% of the total variation in the PCoA plot. The significant correlation of allele frequencies at divergent loci with environmental variables suggested that these populations are adapted to local conditions. Only 17 of 48 wing geometry and standard morphometric parameters were useful for clustering A.m. capensis, A.m. scutellata, and hybrid individuals. CONCLUSIONS: We produced a minimal set of 83 SNP loci and 17 wing geometry and standard morphometric parameters useful for identifying the two RSA honey bee subspecies by genotype and phenotype. We found that genes involved in neurology/behavior and development/growth are the most prominent heritable traits evolved in the functional evolution of honey bee populations in RSA. These findings provide a starting point for understanding the functional basis of morphological differentiations and ecological adaptations of the two honey bee subspecies in RSA.

Phylogeny, new generic-level classification, and historical biogeography of the Eucera complex (Hymenoptera: Apidae).

The longhorn bee tribe Eucerini (Hymenoptera: Apidae) is a diverse, widely distributed group of solitary bees that includes important pollinators of both wild and agricultural plants. About half of the species in the tribe are currently assigned to the genus Eucera and to a few other related genera. In this large genus complex, comprising ca. 390 species, the boundaries between genera remain ambiguous due to morphological intergradation among taxa. Using ca. 6700 aligned nucleotide sites from six gene fragments, 120 morphological characters, and more than 100 taxa, we present the first comprehensive molecular, morphological, and combined phylogenetic analyses of the 'Eucera complex'. The revised generic classification that we propose is congruent with our phylogeny and maximizes both generic stability and ease of identification. Under this new classification most generic names are synonymized under an expanded genus Eucera. Thus, Tetralonia, Peponapis, Xenoglossa, Cemolobus, and Syntrichalonia are reduced to subgeneric rank within Eucera, and Synhalonia is retained as a subgenus of Eucera. Xenoglossodes is reestablished as a valid subgenus of Eucera while Tetraloniella is synonymized with Tetralonia and Cubitalia with Eucera. In contrast, we suggest that the venusta-group of species, currently placed in the subgenus Synhalonia, should be recognized as a new genus. Our results demonstrate the need to evaluate convergent loss or gain of important diagnostic traits to minimize the use of potentially homoplasious characters when establishing classifications. Lastly, we show that the Eucera complex originated in the Nearctic region in the late Oligocene, and dispersed twice into the Old World. The first dispersal event likely occurred 24.2-16.6 mya at a base of a clade of summer-active bees restricted to warm region of the Old World, and the second 13.9-12.3 mya at the base of a clade of spring-active bees found in cooler regions of the Holarctic. Our results further highlight the role of Beringia as a climate-regulated corridor for bees.

Nesting Biology of Xylocopa xinjiangensis (Hymenoptera: Apidae: Xylocopinae).

Xylocopa, an important genus in Hymenoptera: Apidae, is of great significance in research on the early stages of insect social evolution. Most species in this genus burrow into wooden structures. Only the Proxylocopa subgenus nests in the soil. Here, we report the nesting behavior of Xylocopa xinjiangensis (Hymenoptera: Apidae: Xylocopinae), which is distributed only in Western China. During July 2013 and August 2016, we observed the nest architecture and nest building process of X. xinjiangensis. X. xinjiangensis is solitary and nests in the soil walls of gullies, mounds, and cliffs in the Manas area, Xinjiang, multiplying at the rate of one generation a year. Newly emerged females eclose in the fall and build wintering nests first. The next spring, outbound wintering females build breeding nests, although a few wintering females may use the breeding nests built by their mothers. The location and structure of X. xinjiangensis wintering nests are different from those of the breeding nests. The wintering nest is simple in structure, consisting of a tunnel leading perpendicularly from the surface to the interior. The structure of the breeding nest may be either a branching tunnel or a straight-chain tunnel. The first cell that X. xinjiangensis builds in the breeding nest is closest to the entrance, which is a significant difference from the behavior of carpenter bees that construct nests in wood structures. The results of this study lay the foundation for the utilization and protection of X. xinjiangensis resources and facilitate a better understanding of the evolution of the Xylocopa population.