How honey bee drones acquire bacteria in their gut and reproductive tissues
The western honey bee (Apis mellifera) is an important pollinator of crops worldwide. Worker bees forage and perform the tasks that sustain the colony. Drones, the males of the species, contribute genetically by mating with virgin queens from other colonies. Their reproductive fitness can affect queen performance and, in turn, colony survival.
Beekeepers have reported substantial colony losses in the United States and elsewhere. The study cites annual U.S. losses as high as 50.8%. That figure refers to colony losses from all reported causes. Colony collapse disorder (CCD), widely reported from 2007 onward, describes a particular pattern of loss. Pathogens, parasites, poor nutrition, climate change and agricultural chemicals are among the factors investigated in relation to colony health. Queen health is also an important factor in colony survival.
Honey bees are eusocial insects, and a single queen normally produces the colony’s brood. The study cites a historical lifespan of two to seven years in the queen’s original colony and reports that queens are now replaced more often. In the cited studies, about half of colonies replaced a queen within six months and queen replacement was associated with a threefold increase in the risk of colony loss. Workers make up 90% or more of the colony and forage, maintain the hive and care for the queen, drones and newly emerged workers. They may replace a queen for several reasons, including excessive laying of unfertilised eggs, which develop into drones, if the queen’s sperm reserves are depleted or of poor quality.
Drones make up less than 10% of the colony population during spring and summer. Their reproductive role is to mate with virgin queens from other colonies. Their reproductive fitness can therefore affect future queens and the colonies those queens head. Drone health and fertility have received less research attention than worker and queen health.
Drones develop for about 24 days before emerging as adults. Spermatogenesis is completed by emergence, when the seminal vesicles and mucus glands are still developing and sperm remain in the testes (Figure 1). Sperm migrate to the seminal vesicles about six to twelve days after emergence and are stored there until mating. Once migration is complete, the drone is sexually mature and the testes degenerate and cease to function (Figure 1).
Honey bee queens mate early in life, during one or a few mating flights. Colony growth and longevity depend in part on the quantity and quality of sperm collected on those flights. Seminal fluid must help maintain sperm viability in the male’s seminal vesicles, during mating and subsequently in the queen’s spermatheca. Queens rely on stored sperm for the rest of their lives. Drone sperm counts vary widely, but the factors that determine drone reproductive fitness remain incompletely understood.
The gut microbiota contributes to worker honey bee health. Research has linked it to immunity, defence against pathogens, metabolism and potentially behaviour and development. Workers are the largest group in the colony, and their microbiota has been studied extensively. They acquire gut microbes through social interactions and exposure to hive materials. Under typical conditions, the worker gut community is established about five days after emergence and stays relatively stable in healthy bees. It consists mainly of five bacterial groups: Bifidobacterium, Snodgrassella, Gilliamella, Lactobacillus and Bombilactobacillus.These core groups are shared among workers, but the strains within them vary among individuals. This can give different communities different functional capacities.
The drone gut contains some of the same bacterial groups as the worker gut, but is generally less diverse and varies more among individuals.At the time of the study, little was known about how drones acquire their microbiota or whether social interactions help establish it. Microbes have been found in the reproductive tissues of several animals, including insects. In some insects, such as mosquitoes and tsetse flies, reproductive tissue-associated bacteria have been linked to development or reproductive fitness. Such findings motivated the authors to ask whether honey bee reproductive tissues also harbour bacteria and whether these might be relevant to health and fertility.
Queens are difficult to study experimentally because of their indispensable role in the colony. Since a queen’s reproductive fitness and lifespan depend in part on the drones she mates with, drones offer a way to investigate whether bacteria are present in honey bee reproductive tissues and what their role might be.
This study asked whether bacteria occur in drone reproductive tissues, how drones acquire their gut and reproductive tissue-associated microbiota, and how those communities change during maturation. The authors used 16S rRNA gene amplicon sequencing to identify and characterise bacteria in the guts and reproductive tissues of conventionally reared and experimentally reared drones. In particular, the study reported the first detection of bacteria in the reproductive organs of honey bee drones. Drones that emerged naturally and were then kept under sterile laboratory conditions without social contact rarely harboured the bacterial groups found in conventionally reared drones and workers, such as Snodgrassella, Bifidobacterium, Gilliamella, Lactobacillus and Bombilactobacillus. The findings suggest that social contact with workers , for example through feeding and grooming, helps establish the gut and reproductive tissue-associated microbiota of drones. The discovery of a conserved bacterial community in drone reproductive organs also makes the honey bee a candidate model for investigating how microbes associated with reproductive tissues may affect host fitness.
Materials and methods Read the full article
Results
The authors detected a microbiota associated with drone reproductive tissues. This finding opens a line of investigation into drone reproductive fitness. The experiment also indicated that the establishment of typical bacterial communities in the gut and reproductive tissues depended strongly on social interactions with workers, because drones that emerged on hive frames but were kept in the laboratory without worker contact did not develop the typical microbiota. Drones returned to the colony were housed in foster cages with limited contact with hive material. This supports the interpretation that contact with the workers caring for them helped establish their microbiota. As reported previously, drone gut communities were less diverse than worker gut communities and were dominated by Lactobacillus species. This difference may be related to the fact that nurse bees feed and care for drones and often give them less attention when resources are scarce.
The authors hypothesise that the amount of social contact a drone receives during and after emergence influences how its microbiota becomes established. Diet and lifestyle may also account for differences between workers and drones. Workers consume substantially more pollen than drones, and their diet changes as they age and move from nursing to foraging. Foragers spend much of their time outside the hive, whereas drones leave the colony mainly for mating-related flights.
The 16S copy numbers initially suggested unexpectedly high bacterial loads in the guts and reproductive tissues of laboratory-kept drones (NE-D0 and Lab-D5), although the communities lacked typical honey bee-associated groups. One possible explanation is that resident bee-associated bacteria normally outcompete environmental microbes. In the hive, nurse bees help drones remove the wax capping at emergence and feed them immediately, potentially beginning microbiota acquisition. The authors suggest that some or all of the bacteria found in the laboratory-reared drones may have come from their brood cells during emergence. Brood cells can contain plant-derived material and environmental microbes.
Many NE-D0 samples did not yield usable sequencing data, possibly because their guts and reproductive tissues contained very few bacteria. After quality filtering, more than 10% of the reads from the guts of laboratory-kept drones were assigned to mitochondria or chloroplasts or remained unassigned. In some individuals, these reads accounted for more than 40% of the total. This suggests that few bacterial sequences were present. Mitochondrial and chloroplast reads sometimes occur in honey bee gut 16S surveys because host tissue and plant material are present, but they usually account for a small fraction, for example less than 1%. An unusually high fraction of reads from the testes of laboratory-kept drones, particularly NE-D0 drones, also came from mitochondria or could not be assigned. Immature drone testes contain many spermatozoa, whose middle region is largely made up of mitochondrial derivatives.
The 16S copy numbers measured by quantitative PCR (qPCR) in tissues of laboratory-kept drones may therefore overestimate bacterial abundance because the primers can amplify mitochondrial or chloroplast DNA. After the study, the authors found that their qPCR primers were particularly prone to off-target mitochondrial amplification in samples with few bacteria. Gut and reproductive tissue communities were broadly similar in Hive-D5 and Hive-D18 drones, both dominated by Lactobacillus. Differences in total bacterial abundance and relative taxon abundance could reflect shifts during maturation. The authors did not quantify the absolute abundance of individual taxa, so a change in relative abundance does not establish a change in the actual number of bacteria of a given taxon.
The NE-D0, Lab-D5 and Hive-D5 groups were sampled in late summer 2021. Hive-D18 drones came from a different colony in spring 2022, because in summer 2021 the workers stopped tending the drones and the males died before reaching maturity. Previous worker studies found little effect of colony origin on gut community composition but did report seasonal differences in composition and absolute abundance. Winter workers, for example, had higher bacterial abundance and lower within-sample diversity. Season may therefore contribute to the differences between Hive-D5 and Hive-D18. Bacterial loads were higher in spring Hive-D18 drones than in late-summer Hive-D5 drones. The groups showed no significant difference in alpha diversity in the gut or reproductive tissues, and Lactobacillus predominated in both.
Further work is needed to determine how season, colony origin and other environmental factors affect drone microbiota. Gut and reproductive tissue communities contained similar bacterial groups. Sterile dissection, care to keep the gut intact and the substantial bacterial signals in reproductive tissues argue against accidental gut contamination during sampling. Similarities between these communities have also been reported in other insects, including the tomato leafminer (Tuta absoluta) and some mosquitoes.
Honey bees have specialised, coevolved gut communities. Some core bacterial groups may contain strains adapted to particular tissues, with functional differences among them. How bacteria reach the reproductive tissues remains unclear. Given the taxonomic similarity within individual drones, the authors hypothesise that the immune system and the gut barrier are still developing at emergence, potentially allowing bacteria from the gut to reach other organs. Fluorescence in situ hybridisation (FISH) or other fluorescent labelling could help locate cells in the tissues, and metagenomic sequencing and culture could test for strain-level differences.
Worker gut microbes contribute to metabolism, defence against pathogens and immunity, and may also affect development. The effects of gut microbes on drone health were not tested directly, although similarities with worker communities suggest some functions may be shared. This was the first study the authors knew of to characterise bacteria associated with honey bee reproductive tissues. Effects on drone health or fertility, and any subsequent effects on queens and colonies, remain hypotheses.
Future work should determine what the reproductive tissue-associated bacteria do. The study identifies them as a factor to investigate in drone health and reproductive fitness, and suggests that honey bees may serve as a model for research on reproductive as well as gut microbiota.
Published online 19 December 2024 · mSphere · DOI 10.1128/msphere.00705-24
Authors · Alexis Burks (Department of Biology, University of North Carolina, Greensboro, North Carolina, USA) Patrick Gallagher (Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, USA) Kasie Raymann (Department of Biology, University of North Carolina, Greensboro, North Carolina, USA) and (Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, USA)
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