Abstract Sperm competition, as the last form of male competition prior to fertilization, is an important component in the reproductive and evolutionary biology of polyandrous species. Golden cuttlefish (Sepia esculenta) is a commercially important annual cephalopod, in which females often mate with multiple males during their mating season and maintain viable sperm in their sperm storage organs for a long time. This multiple-mating system leads to male competition and ultimately sperm competition. To explore the mechanisms of sperm competition in cuttlefish, a laboratory study was conducted using three experimental approaches. First, two similar-sized males were introduced to sequentially mate with a single female (experiment 1), to investigate mating order effects on paternity. Thereafter, a four-male experiment was run (experiment 2), which again tested for mating sequence on paternal contribution. Finally, to test whether male size is a factor affecting paternity, we compared the paternity of two different-sized males that were sequentially mated with a single female (experiment 3). The paternity of the offspring was analyzed using six microsatellite DNA markers on fertilized eggs. The results revealed last-male sperm precedence in all experiments, and no effect of male size was detected. For the two-male experiment, the last male to mate had a progeny share (P2) of 42.86%–61.90%. For the four-male experiment, the last mated male had the highest progeny share (P4) with a proportion of 35.56%–43.75%. Even with differential male sizes in the two-male experiment, the last mated male retained the highest progeny share (P2, 58.33%–91.67%). This study shows a consistent pattern of last-sperm advantage across different experiments and contributes to growing research on cephalopod mating systems. Proper mating schemes should be considered during seedling production for S. esculenta to ascertain efficient resource use and genetic diversity in produced offspring.

中文翻译：

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金乌贼Sepia esculenta的精子竞争：交配顺序和雄性大小的影响

摘要 精子竞争是受精前雄性竞争的最后一种形式，是多雄性物种生殖和进化生物学的重要组成部分。金乌贼 (Sepia esculenta) 是一种具有重要商业价值的一年生头足类动物，雌性在交配季节经常与多只雄性交配，并在精子储存器官中长时间保持有活力的精子。这种多重交配系统导致雄性竞争，最终导致精子竞争。为了探索墨鱼精子竞争的机制，使用三种实验方法进行了一项实验室研究。首先，引入两个体型相似的雄性，依次与单个雌性交配（实验 1），以研究交配顺序对亲子关系的影响。此后，进行了四名男性实验（实验 2），这再次测试了父系贡献的交配序列。最后，为了测试雄性体型是否是影响亲子关系的因素，我们比较了两个不同体型的雄性依次与单个雌性交配的亲子关系（实验 3）。使用受精卵上的六个微卫星 DNA 标记分析后代的亲子关系。结果显示在所有实验中最后一个男性精子优先，并且没有检测到男性大小的影响。对于双雄性实验，最后交配的雄性的后代份额 (P2) 为 42.86%–61.90%。对于四雄性实验，最后交配的雄性后代比例最高（P4），比例为 35.56%–43.75%。即使在两雄性实验中雄性大小不同，最后交配的雄性仍保留最高的后代份额（P2，58.33%–91.67%）。这项研究显示了不同实验中最后精子优势的一致模式，并有助于对头足类交配系统的研究不断增长。在 S. esculenta 的幼苗生产期间应考虑适当的交配方案，以确定所生产后代的有效资源利用和遗传多样性。