PDF《Fluorescent sperm offer a method》

1 Fluorescent sperm offer a method for tracking the real-time success of ejaculates when they compete to fertilise eggs Rowan?A.

?Lymbery,W.?Jason?Kennington &

Jonathan?P.?Evans Despite intensive research effort, many uncertainties remain in the field of gamete-level sexual selection, particularly in understanding how sperm from different males interact when competing for fertilisations. Here, we demonstrate the utility of broadcast spawning marine invertebrates for unravelling these mysteries, highlighting their mode of reproduction and, in some species, unusual patterns of mitochondrial inheritance.We present a method utilising both properties in the blue mussel, Mytilus galloprovincialis. In mytilids and many other bivalves, both sperm and egg mitochondria are inherited.We exploit this, using the vital mitochondrial dye MitoTracker, to track the success of sperm from individual males when they compete with those from rivals to fertilise eggs.We confirm that dying mitochondria has no adverse effects on in vitro measures of sperm motility (reflecting mitochondrial energetics) or sperm competitive fertilisation success.Therefore, we propose the technique as a powerful and logistically tractable tool for sperm competition studies. Importantly, our method allows the competitive fertilisation success of sperm from any male to be measured directly and disentangled from confounding effects of post-fertilisation embryo survival. Moreover, the mitochondrial dye has broader applications in taxa without paternal mitochondrial inheritance, for example by tracking the dynamics of competing ejaculates prior to fertilisation. Darwin1 first proposed sexual selection as an evolutionary force acting on variation in reproductive success caused by (1) intrasexual competition for mates (typically among males), and (2) intersexual mate choice (typ- ically females choosing preferred males). Since then, sexual selection has become a major focus of evolutionary and behavioural research2 . A critical turning point in the field of sexual selection was the recognition that females often mate with multiple males, or their eggs are exposed to sperm from multiple males, meaning that sexual selection can continue after gamete release3 . This occurs as sperm competition, where ejaculates from rival males compete for fertilisations4 , and cryptic female choice, where females influence the outcome of such contests5,6 . Both of these mechanisms of sexual selection are widespread across most sexually reproducing taxa and consti- tute important evolutionary forces acting on both sexes7C9 . Although these processes are commonly termed '

post- copulatory sexual selection'

10 , we prefer the term '

gamete-level sexual selection'

to include externally fertilising animals that do not pair or copulate. Despite intensive research on gamete-level sexual selection, there remains a taxonomic bias toward mobile, terrestrial and internally fertilising animals3,11,12 . In particular, relatively few studies have focused on broadcast spawning marine invertebrates. Typically, these animals have sedentary or sessile lifestyles and both sexes release gametes directly into the ocean, where fertilisation occurs13 . Although largely neglected in the context of sex- ual selection (but see refs 11,14), broadcast spawners exhibit several attributes that make them ideally suited for understanding gamete-level sexual selection. First, the absence of mating competition or mate choice prior to gamete release means that sexual selection operates exclusively through gamete-level interactions11 . Second, broadcast spawners offer highly tractable systems for controlled in vitro experiments on gamete-level interactions. This tractability has been utilised in recent studies to characterise patterns of multivariate selection on gam- etes15,16 , examine variation in male-female gametic and genetic compatibilities17,18 , and explore the transmission Centre for Evolutionary Biology, School of Animal Biology, University of Western Australia, Crawley 6009, WA, Australia. Correspondence and requests for materials should be addressed to R.A.L. (email: rowan.lymbery@ research.uwa.edu.au) received: