Everyone knows honeybee females (queens) mate at the beginning of their adult life and are then unable to mate again. A queen mates with many males (drones), often on a single occasion but sometimes after multiple flights in successive days. The mating is very quick, not more than 5 seconds and perhaps no more than one or two seconds, after which the male is paralysed and dies.
Competition between males in a mating congregation occurs, mostly as a result of size and power, and some selection operates seemingly on the basis of flight altitude, different strains favouring different heights. A single drone congregation area might contain more than 20,000 drones from potentially hundreds of colonies, and the chance of an individual male being able to mate more than once would be very low. In honeybees therefore it’s not surprising a male expends his entire effort mating with a single queen, and not surprising that probably his best chance of improving his reproductive success is posthumous sperm competition.
Queens, at least during mating, appear to have very little ability to choose the paternity of her offspring, but there are good reasons to suppose her interest is in being able to produce a sizeable range of genetic characteristics. Variation is good for managing conflict in a social group, protecting the colony from diseases and environmental change, and providing progressive, adaptable worker performance. Especially in honeybees, as she can never mate again, she has a particular interest in actually opposing or counteracting individual males’ reproductive success, negating sperm competition and choosing diversity. This ‘choice’ is said to be ‘cryptic’, because it is hidden from the male (Eberhard, 1996).
Queens are estimated to lay about 200,000 eggs each year; something like 1.0 – 1.6 million fertilised eggs in her 5 – 8 year lifetime. Some hymenoptera (species of ants) do better by a significant margin, producing 8 million workers fertilised by sperm stored for decades.
Much more sperm are stored in the spermatheca (in the order of 5 million) than will be needed. Drones will produce between 2 million and 12 million spermatozoa each, and at the end of a mating flight a queen might contain 200 million or so temporarily housed in her oviducts, vagina, and bursa copulatrix. Only around 2.5% of the sperm she acquires during mating is stored, and even less are actually used (think an average two per egg over her lifetime). Spermatozoa can be stored for many years and retain viability. Paternity studies have shown it is completely mixed and used equitably.
Discovered in 1905 the key to this remarkable economy of sperm use is something called a Bresslau sperm pump. This structure sits between the spermathecal and the spermathecal duct, a valve in muscular tissue that, if you like, ‘reaches in’ and grabs a constant volume of spermathecal fluid (containing sperm) and transports it out to the eggs. (After mating it ‘pumps’ in the opposite way, filling the spermatheca). While the fluid volume is replaced and always stays the same, the density of spermatozoa it contains gradually declines. The Bresslau sperm pump is also found in ants. With the instruments available nowadays it’s actually possible to count sperm on eggs.
Just how bees, wasps and any are able to keep spermatozoa alive for so long eludes a complete explanation, but in short, by an extreme conservation of energy and reduction in oxidative stress. Both seminal fluid and spermathecal fluid must have a role in providing a habitat that nourishing the cells, reduces oxidative stress, and protects them from pathogens, but it’s most likely spermathecal fluid evolved to maximise their long-term viability. Studying spermathecal fluid from virgins and mated queens shows they do differ, but also have some functional similarity with some elements in seminal fluid. Drones too store sperm, although not for as long. In a process that takes at least 40 hours it appears that the storage ‘environment’ is gradually changed from semen to a receptive queen’s spermathecal fluid, to a mated queen’s spermatheca. Spermatozoa in the spermathecal fluid ‘acclimatise’ to their new environment and begin to metabolise very, very slowly, essentially ‘outsourcing’ some of their vital functions to the female environment. In particular, while spermatozoa are able to metabolise aerobically, in storage there is evidence to suggest they switch to anaerobic energy production using a partly metabolised product in spermathecal fluid to limit the release of damaging Reactive Oxygen Species (ROS). As well, the spermatheca is a bead-like organ with two spermathecal glands situated outside a hard sclerotized wall impervious to oxygen. By comparison to other organs the spermatheca has significantly lower oxygen concentrations inside. Spermathecal fluid is also known to contain many highly-active antioxidant enzymes, and these increase if we compare virgins with mated queens.
It's become well documented in many species that males don't just transfer spermatozoa during copulation but include a complex mixture of molecules, anti-oxidants, ions and cells other than spermatozoa, including sometimes pathogenic micro-organisms. These male compounds have a variety of functions. Some directly affect the sperm’s survival in the female’s reproductive tract, providing nutrition, pH and osmotic buffering, and defences against oxidizing agents. Other products have important effects on the physiology and behaviour of the female, such as promotion of sperm transport, and inducing ovulation or oviposition. We are now beginning to realise that seminal fluid contains molecules that have a demonstrable effect on gene expression, and that a number of proteins cross the vaginal wall into haemolymph where they can bind to receptors on neurons directly affecting nerve signalling.
A somewhat surprising example, consistent with other insect studies and earlier work, establishes a (short-term) loss of visual ability in queens linked to a peptide transferred in male semen. The effect is that queens are less inclined to undertake further mating flights (because they can’t see properly), but with the consequence that the queen tries to fly earlier if she can, before the loss becomes too debilitating. Males have no interest in queens flying to mate with more males. The scientists used RNA-sequencing to look at the changes in gene expression following artificial insemination, comparing them with naturally inseminated queens and queens inseminated with a saline control. They were able to identify the changed genes as ones known to be associated with functions that mostly enable vision. They then carried out a similar exercise, but this time measured the actual performance of the eyes (all of them!), things like their response to different light frequencies, and sensitivity to visual contrast. Last, they used RFID tags to monitor natural flight activity (and queen loss) after the same set of treatments (insemination, mock insemination etc). Each set of experiments indicated that queen’s visual performance deteriorated 24 – 48 hrs after receiving seminal fluid, and they were more likely to be lost on subsequent mating flights. The same effect has been observed in other studies of fruit flies, a parasitoid wasp, and in the bumble bee Bombus terrestris, suggesting that this ability to manipulate female mating using components of seminal fluid could be widespread or even universal amongst Hymenoptera and perhaps all insects.
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