Ant queens mate on a single occasion early in life and store millions of sperm cells in their spermatheca. By carefully using stored sperm to fertilize eggs, they can produce large colonies of thousands of individuals. Queens can live for decades and their lifetime reproductive success is dependent on their ability to keep stored sperm alive. Maintaining high sperm viability requires metabolic energy which could trade-off with other costly processes such as immunity. We tested the impact of immune activation on the survival of stored sperm by prompting Lasius niger ant queens to mount a melanization response and subsequently measuring sperm viability in their spermatheca. Since queens face different challenges that influence energy allocation depending on the life stage of their colony, we measured sperm viability after immune activation in both newly mated queens (incipient) and in queens 1 year after mating (established). We found that immune activation reduced sperm viability in established queens but not in incipient queens, showing that the cost of immunity on sperm preservation depends on the life stage. Unexpectedly, established queens had significantly higher sperm viability in their spermatheca compared to incipient queens suggesting that ant queens are able to remove dead sperm from their spermatheca.

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Leaf-cutting ant queens mate with multiple males during a single nuptial flight and store sperm for up to two decades. During mating, males transfer sperm from their accessory testes to the queen bursa copulatrix from where it enters the spermatheca, an insect sperm storage organ that has become highly specialized in long-lived ant queens who never re-mate later in life. Long-term storage without the possibility to obtain new sperm creates an immune defence dilemma, because recognition of non-self cells eliminates infections but may also target irreplaceable sperm and reduce lifetime reproductive success. We therefore hypothesized that non-specific immune responses, like pathogen melanization, should be silenced in the spermatheca, because they rely on general non-self recognition, and that specific responses such as antimicrobial peptides are activated instead as they specifically target pathogenic bacteria and/or fungi. The maintenance of uninfected sperm cells by males before mating is not constrained by non-self recognition, meaning immune regulation might be more liberal in male reproductive organs. To test this hypothesis, we measured gene expression of two antimicrobial peptides, abaecin and defensin, and prophenoloxidase, an important enzyme of the melanization pathway, in male accessory glands and testes and in queen bursae copulatrix and spermathecae of and leaf-cutting ants. As expected, prophenoloxidase expression was low in reproductive organs that sustain prolonged contact with sperm, whereas antimicrobial peptides showed average to high expression, indicating that leaf-cutting ants invest in specific rather than generalist immune defences for pathogen protection in organs that store sperm.

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