Abstract
Honeybee males produce ejaculates consisting of large numbers of high quality sperm. Because queens never re-mate after a single mating episode early in life, sperm are stored in a specialised organ for years but the proximate mechanisms underlying this key physiological adaptation are unknown. We quantified energy metabolism in honeybee sperm and show that the glycolytic metabolite glyceraldehyde-3-phosphate (GA3P) is a key substrate for honeybee sperm survival and energy production. This reliance on non-aerobic energy metabolism in stored sperm was further supported by our findings of very low levels of oxygen inside the spermatheca. Expression of GA3P dehydrogenase (GAPDH), the enzyme involved in catabolism of GA3P, was significantly higher in stored compared to ejaculated sperm. Therefore, long-term sperm storage seems facilitated by the maintenance of non-aerobic energy production, the need for only the ATP-producing steps of glycolysis and by avoiding sperm damage resulting from ROS production. We also confirm that honeybee sperm is capable of aerobic metabolism, which predominates in ejaculated sperm while they compete for access to the spermatheca, but is suppressed during storage. Consequently, the remarkable reproductive traits of honeybees are proximately achieved by differential usage of energy production pathways to maximise competitiveness and minimise damage of sperm.
Highlights
Honeybee males produce ejaculates consisting of large numbers of high quality sperm
GA3P use yielded a significantly higher ATP production via acidifying glycolytic metabolism when compared to glucose or fructose use (Mann-Whitney nonparametric U test: U = 50.0; nGlucose = 15, nGA3P = 15; p = 0.013 and U = 53.0; nFructose = 15, nGA3P = 15; p = 0.009, Fig. 1B). This increase in ATP production was driven by the higher rate of acidifying glycolytic rate for GA3P compared with glucose or fructose (Supplementary Fig. 1)
When we independently quantified the effect of GA3P and fructose on sperm survival, we found that sperm viability was significantly higher in samples containing GA3P compared to fructose or the control treatment without any substrate (Wilcoxon Signed-Rank test; n = 10; Z =−2.934; p < 0.001, Fig. 2)
Summary
Honeybee males produce ejaculates consisting of large numbers of high quality sperm. Because queens never re-mate after a single mating episode early in life, sperm are stored in a specialised organ for years but the proximate mechanisms underlying this key physiological adaptation are unknown. Protein abundance differs between ejaculated and stored honeybee sperm, including a number of glycolytic enzymes, providing further evidence that energy metabolism is key for their long-term survival[17]. Glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enolase, were more abundant in stored sperm but had a lower activity, suggesting wear and tear during storage[17]. Glycolytic enzymes are prominent in the female-derived spermathecal fluid but absent in male-derived seminal fluid[20] Based on these earlier studies, we quantified the effects of different substrates on energy metabolism, sperm physiology and survival. Oxygen concentrations inside the spermatheca were measured and shown to be substantially lower than in other honeybee tissues investigated
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