ORIGINAL ARTICLE Effects of female mating status on copulation behaviour and sperm expenditure in the freshwater crayfish Austropotamobius italicus Paolo Galeotti & Fabio Pupin & Diego Rubolini & Roberto Sacchi & Pietro A. Nardi & Mauro Fasola Received: 4 August 2006 / Revised: 5 September 2006 / Accepted: 26 October 2006 / Published online: 18 November 2006 # Springer-Verlag 2006 Abstract Sperm competition models predict that males should adjust their sperm expenditure according to the risk and/or intensity of sperm competition. In this paper, we analysed copulatory behaviour of both sexes and sperm expenditure in relation to female mating status (virgin or mated) in the freshwater crayfish Austropotamobius itali- cus, a species where males have been reported to feed on and remove sperm laid by other males. The same females were allowed to be inseminated sequentially by two males, and we compared the sexual behaviours of partners between the first (virgin females) and the second mating (mated females). We found that female resistance did not differ between the first and the second mating, nor males refused or took more time to mount a mated female. However, when mating with a mated female, males reached an effective copulation position significantly later. This occurred because second-mating males removed, by eating, all or most spermatophores previously deposited by first males. As removal was often incomplete, this resulted in a larger amount of sperm being deposited on female ventral parts after the second mating, although second males did not allocate more sperm to mated females than first males did. Thus, the peculiar mode of sperm competition, where males remove previously deposited sperm, and the conse- quent predictable strong last male prevalence in paternity likely led to the observed lack of adjustment of sperm expenditure to female mating status in this species. Keywords Copulation . Crustaceans . Multiple mating . Sperm competition . Sperm expenditure Introduction In polyandrous species, where both sexes mate multiply, male fertilization success may often depend on the ability of his sperm to successfully compete with that of his rivals (Simmons and Siva-Jothy 1998; Simmons 2001). Sperm competition, which is defined as the post-copulatory competition between the sperms of two or more males to fertilize female eggs during a breeding season (Birkhead and Møller 1992, 1998; Parker 1998), results inevitably in an evolutionary arms race (Dawkins and Krebs 1979) between mechanisms of pre-emptive advantage in compe- tition for fertilizations and counter-mechanisms aimed at preventing preemption (Parker 1984). Such arms races have produced a variety of morphological, behavioural and physiological adaptations enhancing the competitive ad- vantage of a male’ s sperm or countering the sperm of competitors (Smith 1984). The adaptations that arise in particular cases depend on several factors, e.g. the morphology of the female reproductive tract, the existence and form of sperm storage organs and the opportunity for multiple mating (Birkhead and Møller 1992). Recent models suggest that males should increase their ejaculate expenditure when experiencing a high risk of sperm competition (see Wedell and Cook 1999; Wedell et al. 2002). For example, the mating status (virgin or mated) of a polyandrous female may alter her reproductive value to a male, and males should be able to assess it and vary their reproductive behaviour accordingly, e.g. by increasing sperm number or displacing rival sperm when mating with a mated female. In most instances, virgin females are preferred as mates, as they offer a reduced probability of Behav Ecol Sociobiol (2007) 61:711–718 DOI 10.1007/s00265-006-0301-2 Communicated by P. Backwell P. Galeotti (*) : F. Pupin : D. Rubolini : R. Sacchi : P. A. Nardi : M. Fasola Laboratorio di Eco-Etologia, Dipartimento di Biologia Animale, Università degli Studi di Pavia, p.zza Botta 9, 27100 Pavia, Italy e-mail: galeozot@unipv.it