Photo: Alison McAfee
Honey bee queens keep drones’ sperm alive inside them for years, but some surprising environmental triggers can cause massive sperm death.

“Give the drone’s thorax a pinch,” Jeff Pettis instructed as I took the drone he handed me. “Then squeeze the abdomen between your thumb and forefinger, rolling from the front of the abdomen to the tip.” I flipped the drone upside down and started to pinch the thorax as coached, feeling a crispy crunch as it buckled and killed the bee. I stifled a shudder – I always hate the sensation of crushing an exoskeleton, whether it belongs to a bee or another insect. The drone’s abdomen still pulsed with reflexive breaths, which quickly stopped as I squeezed it from front to back.

Jeff was showing me how to harvest fresh semen from drones. That day, we were just practicing, but semen collected in this way can then be used for instrumental insemination or tested for sperm viability. And sperm viability is a critical determinant of queen quality, and ultimately, colony longevity—dud sperm directly translate into fewer workers, less genetic diversity, and potentially smaller colonies. New research suggests that sperm viability is compromised by common challenges like temperature stress and pesticide exposure, even more than previously thought.

As I squeezed the abdomen, it felt like it was going to explode between my fingers. But sure enough, instead of bursting, the endophallus shot out from the hind end, pausing with two bright orange horns (cornuae) poking out as if someone was blowing into an inside-out rubber glove. It looked truly alien. “That’s a partial eversion,” Jeff confirmed. “Now keep squeezing.”

Figure 1
Drones have elaborately structured penises. A) A drone awaiting penile eversion. B) Partial eversion, with the corunuae clearly visible (Y-shaped horns). They are thought to act like hooks, helping to grip the queen during mating. C) Full eversion, displaying the whole endophallus. Photos by Alison McAfee

The rest of the phallus exploded from the abdomen with a pop. Sitting on top of the bulbous end were a few microlitres of creamy café au lait colored semen, poised for collection.

A drone’s purpose in life is to grow big enough and strong enough to compete with other drones for access to a virgin queen during her nuptial flight. If successful, they mate in mid-air, he deposits that drop of semen inside of her (explosive pop included), his phallus rips from his body, and he falls to his death. For every queen, about fifteen to twenty drones mate with her and reach this morbid fate. But while the drones themselves may die, their sperm will live inside the queen for years, fathering millions of offspring over the course of the queen’s life.

How is it possible for sperm to stay alive in the queen for so long, when in other animals, they live only minutes or days? Human sperm, for example, only stays alive outside a male’s body for about twenty minutes (or several days, in the female’s fallopian tube). Queen ants, which are also social insects, put even honey bees to shame; most ant queens store live sperm for upwards of ten years.

In a social insect’s kingdom, the dogma is not only long live the queen, but also long live the queen’s sperm. Researchers have been searching for the mechanism of these remarkable feats of sperm storage since the 1960s, when Eugenia Alumot, from Rehovot, Israel, began investigating the sugars and enzymes that could be providing honey bee sperm with energy inside the queen’s spermatheca (the specialized organ that stores the sperm).

Now, we know that after a burst of energy used to out-swim other sperm and gain access to the spermatheca, the sperm fall into a fairly quiescent life. Specific metabolic pathways are dialed back, so once inside the spermatheca, the sperm metabolize nutrients differently. This probably enables them to minimize damage – and cell death – caused by molecules called “reactive oxygen species,” which are by-products of some types of energy-generating metabolism. But these carefully tuned metabolic shifts can easily be thrown off-balance.

A few years ago, Jeff Pettis and other researchers found that failing colonies tended to be headed by queens with sub-par sperm viability in their spermathecae. Many of the queens’ precious sperm (almost half of their lives’ supply, in fact) were dead. Pettis and colleagues published a paper in PLOS One describing how when they dissected spermathecae and looked at the sperm they contained under a microscope to count how many were alive and dead, the queens heading colonies that were rated as being in “good health” by beekeepers tended to contain sperm with high viability (around 85-92% were still alive). In contrast, queens heading colonies rated as “failing” contained sperm with low viability (around 50-55%). They concluded that sperm viability is linked to colony longevity, but it wasn’t clear what exactly was causing these differences in viability in the first place.

Boris Baer, a professor of entomology at the University of California, Riverside, has made a career out of studying sexual selection in social insects, including honey bees. Years before Pettis et al. published their work, Baer and colleagues found that temperature stress can decrease drone sperm viability. After holding drones at 102.2 oF (39 oC) for four hours, they observed a small but significant decrease in viability from 97 to 90%. 102.2 oF isn’t all that hot, and the researchers would have liked to test a more severe heat shock, but as they noted in their results, “drones are surprisingly heat sensitive. When we exposed males to a temperature of 40 °C for 24h, they all died.” Baer et al. found a similarly small but significant decrease in viability when ejaculated sperm, rather than the drones themselves, were heated. But what if the sperm don’t get a shock of heat within the drone or a test tube, but within the queen herself?

Pettis was intrigued by the idea that temperature spikes could reduce sperm viability inside the queen because there’s one common scenario where queens are exposed to variable temperatures: shipping. When the researchers included temperature loggers in queen shipments, they found that it’s not uncommon for queens to be exposed to temperatures below 46.4 oF (8 oC) or spikes above 107.6 oF (42 oC). They reasoned that this could be one real life situation impacting sperm viability within the queen, and likewise, queen quality. To mimic the temperature extremes that can happen during shipping, they either cold-shocked queens in the lab at 39.2 oF (4 oC), heat-shocked them to 104 oF (40 oC), or kept them at a comfy 86 oC (30 oC) as a control, and tested their sperm viability.

Alarmingly, after being exposed to these temperatures for two hours, the viability of sperm within both heat- and cold-shocked queens was about 60% lower than the controls. This is a far bigger effect than Baer observed in his heat-shocked drones, despite queens being more tolerant to temperature changes themselves. Together, these results suggest that the temperature spikes reduce viability a little by directly stressing the sperm, but a lot by causing some yet-unknown, fundamental change in the queen, likely within her spermatheca. Since metabolism appears to be so important for sperm storage, my guess is that shifts in temperature might cause shifts in metabolism toward the higher energy-producing – but more lethal – state. In my future research, I hope to figure out why these temperature spikes have such a drastic effect.

But temperature stress isn’t the only factor we know that affects sperm viability in the modern world. Imidacloprid, the most commonly used neonicotinoid pesticide, also reduces sperm viability, even at sub-lethal doses. In a different paper published in the Journal of Insect Physiology, Pettis and the rest of his research team found that doses as low as 0.02 ppb of imidacloprid, administered to queens as a topical dab to the abdomen once a day for a week, caused an average of 50% sperm viability reduction compared to controls. While the researchers recognize that the queen is most likely going to come in contact with such a pesticide through the food fed to her by workers, rather than through her abdominal cuticle, they argue that she could also be exposed chronically through contaminated wax. At the University of Bern, Switzerland, other researchers have shown that two other pesticides decrease sperm viability in the drones when fed to colonies in pollen patties, but they did not test the effect on sperm stored in the queens.

These experiments all point to the lesson that we need to protect our queens if we’re going to protect our colonies. We are at the mercy of the shipper when it comes to temperature variation during transport, and queens shipped by US Postal Service and United Parcel Service fare similarly. However, there is one major way we, as beekeepers, can intervene: buy local queens that don’t need to be shipped. Depending on your location and the needs of your operation, this may or may not be possible for you. But if it is, consider going local. It just might help your queens keep their sperm alive, and let your colony thrive.

References:

  1. Keller L. (1998). Queen lifespan and colony characteristics in ants and termites. Insectes Sociaux. 45(3):235-46.
  2. Alumot E, Lensky Y, Holstein P. (1969). Sugars and trehalase in the reproductive organs and hemolymph of the queen and drone honey bees (Apis mellifica var. Ligustica spin.). Comparative Biochemistry and Physiology. 28(3):1419-25.
  3. Paynter EA et al. (2017). Insights into the molecular basis of long-term storage and survival of sperm in the honeybee (Apis mellifera). Scientific Reports. 7: 40236.
  4. Pettis JS et al. (2016). Colony failure linked to low sperm viability in honey bee (Apis mellifera) queens and an exploration of potential causative factors. PLoS ONE.
  5. Stürup M et al. (2013). When every sperm counts: Factors affecting male fertility in the honeybee Apis mellifera. Behavioural Ecology. 24(5):1192-98.
  6. Chaimanee V et al. (2016). Sperm viability and gene expression in honey bee queens (Apis mellifera) following exposure to the neonicotinoid insecticide imidacloprid and the organophosphate acaricide coumaphos. Journal of Insect Physiology. 89:1-8.
  7. Straub L et al. (2016). Neonicotinoid insecticides can serve as inadvertent insect contraceptives. Proceedings of the Royal Society B. 283(1835):1-8.

This article appeared in the August 2018 issue of American Bee Journal.

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