1. Introduction/Background
Insect pollination of commercial crops is valued worldwide at
$175 billion annually and pollination services provided by commercially managed honey bee (
Apis mellifera) colonies in the United States alone are valued at
$14.6 billion annually [
1]. However, commercial beekeepers in the US have reported up to 45% annual colony losses since 2006 [
2,
3,
4,
5,
6,
7,
8,
9]. Multiple factors have been implicated, including agrochemical exposure, forage quality and availability, management practices, parasites, pathogens, and queen reproductive failure [
2,
3,
4,
5,
6,
7,
8,
9]. However, factors specifically impacting queen fertility and their subsequent roles in colony health have received limited attention. In fact, commercial beekeepers have frequently reported queen failure and the ectoparasitic mite
Varroa destructor as the two most common reasons for colony losses in the past several years [
4,
10]. Queen failure occurs when the queen is no longer reproductively fit and she stops efficiently laying eggs or begins laying unfertilized eggs that become drones (male bees) [
11]. While queens can typically live 2–3 years, commercial beekeepers have started replacing queens at least once per year due to poor queen quality and frequent queen failure [
12].
Honey bee colonies are composed of tens of thousands of sterile female workers, hundreds to thousands of seasonal haploid male drones, and a single queen, the only member of the colony that can lay both fertilized and unfertilized eggs [
13]. Roughly a week after emerging, virgin queens undertake one to a few nuptial flights over several days [
13]. Honey bee queens are polyandrous and they mate with multiple drones, which reach sexual maturity about two weeks after emergence [
13,
14,
15,
16,
17,
18,
19,
20]. During a nuptial flight, the queen flies up to 3 km away from her hive to rendezvous with thousands of drones at a drone congregation area (DCA), located 5–40 m above ground [
20]. Older reports have determined that queens mate with an average of 12 drones [
13,
21], but recent work found that queens can mate up to 34–77 drones [
22]. During copulation, the drone irreversibly everts its endophallus into the female, transfers his semen into the oviduct, and drops to the ground to die [
20,
23]. Roughly 10% of each male’s ejaculate is transferred into the queen’s oviduct [
13,
20,
24,
25].
Once a queen has terminated her final nuptial flight and returns to the hive, she starts to store sperm in her spermatheca, a specialized organ found in many insects to facilitate spermatozoa storage, and commences egg laying [
20]. Only about 3% to 5% of ejaculated spermatozoa from each drone is stored in the queen’s spermatheca for future egg fertilization [
13,
20,
24,
25]. A queen can store approximately five to six million total spermatozoa in her spermatheca [
14,
20,
26]. While it varies based on number of stored spermatozoa, queens are highly efficient and fertilize each egg with a median of two spermatozoa; queens that are inseminated with more semen tend to store more spermatozoa and, in turn, fertilize eggs with more spermatozoa [
27]. Honey bees are parthenogenic and queens lay both fertilized eggs that hatch into diploid female workers or queens and unfertilized eggs that develop into haploid drones [
20]. The type of egg that is laid depends on the type of comb cells into which the queen is laying—larger cells are reserved for drones while worker eggs are laid into smaller cells [
20]. In addition to her role as the primary reproductive female in a colony, the queen also continuously releases a blend of pheromones that passively maintain social cohesion of the hive and other aspects of colony organization [
13]. More comprehensive information on the mating biology of honey bees can be found in Reference [
20].
Honey bee queens are typically assessed for their quality based on reproductive longevity, potential amount of viable brood they can produce, the number of the drones with which they have mated, and the genetic diversity of her mates [
28]. There are several traits that are associated with queen quality, including overall weight [
11,
28,
29,
30,
31,
32,
33], weight of the ovaries [
34,
35,
36], weight of the spermatheca, and the number of viable stored spermatozoa [
11,
28,
29,
37,
38,
39,
40]. Queen quality is impacted by the age at which larvae are nutritionally directed into the queen developmental pathway via continued feeding of royal jelly [
12,
41,
42,
43]. Ideally 1st instar larvae are used, but older larvae may be reared into queens if the mother queen is unexpectedly lost [
12,
41,
42,
43]. Queen quality is also affected by genetic background [
29], pesticide exposure [
44,
45,
46], and parasite or pathogen load [
28]. For a more comprehensive review regarding the relationship of these factors to queen quality, see Reference [
28].
Importantly, queen reproduction is also affected by mating conditions [
47,
48,
49,
50,
51,
52,
53]. When a queen mates with drones, she undergoes extensive behavioral, physiological, and molecular changes, including reduced sexual receptivity, ovary development, ovulation, modulation in pheromone production, and transcriptional regulation. These changes contribute to aspects of queen reproductive quality with potential far-reaching implications [
47,
54,
55,
56,
57,
58]. Studies utilizing instrumental insemination have determined that drone semen and seminal fluid, a major component of semen containing numerous proteins and metabolites, initiates many of these post-mating changes in queens and likely plays an important role in shaping queen quality [
47,
48,
49,
50,
51,
52,
53]. However, the specific molecules in semen and seminal fluid involved in initiating post-mating changes in queens have yet to be identified. Hereinto, we review the currently available work investigating post-mating changes in queens and the different copulation factors that influence queen fertility. We then cover recent work identifying the proteins in drone seminal fluid and their potential roles in queen quality and post-mating changes. Furthermore, since queens are polyandrous, they are at greater risk of being infected with sexually transmitted pathogens, such as
Nosema spp. or Deformed Wing virus, which may threaten their health and fitness [
59,
60,
61,
62]. Therefore, we also review research investigating diseases and antimicrobial mechanisms of drone seminal fluid and its ability to reduce parasite transmission during mating. We conclude by discussing future avenues of research.
5. Conclusions and Future Directions
The queen is an important member of the honey bee colony and can be a major determinant of colony health and productivity. Drones, too, are very important players as they can have a strong impact on queen post-mating changes and subsequent colony health. Specifically, drone seminal fluid modulates queen sexual receptivity, pheromone production and seminal fluid proteins are likely the key drivers of these changes [
105,
106]. In order to further solidify the role of SFPs in queen post-reproductive changes, additional studies involving the separation of proteins from the non-protein fraction of seminal fluid and testing their effects on queen post-mating changes are forthcoming. Furthermore, specific SFPs and their functions could be identified via fractionation of proteins (e.g., ion chromatography) and testing their individual effects [
147]. RNAi mediated gene knockdown [
173] or CRISPR-Cas9 gene knockout [
174] of genes encoding SFPs in drones will also likely provide exciting and enlightening paths towards a more holistic understanding of the functions of drone SFPs.
In addition, drone seminal fluid proteomes vary based on genetic lineage [
145]. Based on these differences, future investigations should seek to understand if protein-level differences in honey bee seminal fluid composition due to genetic background (e.g., European versus Africanized bees) result in differential queen post-mating changes and if queens from different genetic lineages exhibit differential post-mating changes.
Furthermore, it is important to reiterate that honey bee seminal fluid is composed of both proteins and unidentified non-protein components, which are likely peptides, lipids, and sugars [
110,
115]. In addition to having antimicrobial activity against
N. apis [
147], the non-protein fraction of seminal fluid may also impact queen post-mating changes and reproduction. In two cricket species,
Teleogryllus commodus and
Acheta domesticus, prostaglandins present in their seminal fluid are responsible for reducing sexual receptivity and inducing oviposition in recipient females [
175,
176]. Prostaglandins are important for honey bee immunity [
175], but it is unknown if they are also present in drone seminal fluid. Thus, more comprehensive studies, including metabolomics or peptidomics approaches, should yield insights into the role of non-protein fractions of seminal fluid in queen health and reproduction.
Lastly, identifying the specific roles of SFPs in queen reproduction could have an important impact on improving bee breeding practices in order to develop more resilient genetic honey bee stock. For example, being able to manipulate the production of specific SFPs in drones could lead to improved queen reproductive fitness particularly in breeder queens. Such selective breeding practices were utilized to develop
V. destructor-resistant honey bees that exhibit higher expression of proteins associated with
V. destructor resistance [
177,
178]. Ultimately, improving and understanding the underlying mechanisms of, and improving drone reproductive health has a great potential to improve resultant queen and colony health and contribute towards reducing colony losses.