Hidden Female Physiological Resistance to Male Accessory Gland Substances in a Simultaneous Hermaphrodite Monica Lodi1,2,* and Joris M
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© 2017. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2017) 220, 1026-1031 doi:10.1242/jeb.149963 RESEARCH ARTICLE Hidden female physiological resistance to male accessory gland substances in a simultaneous hermaphrodite Monica Lodi1,2,* and Joris M. Koene1,2 ABSTRACT sperm for fertilization (the latter being referred to as cryptic female To increase fertilization chances compared with rivals, males are choice) are two evolutionary drivers that are difficult to separate as favoured to transfer accessory gland proteins to females during they both predict differential use of ejaculates in fertilization mating. These substances, by influencing female physiology, cause and favour post-copulatory adaptations that ultimately increase alteration of her sperm usage and remating rate. Simultaneously reproductive success (Eberhard, 1996; Birkhead and Pizzari, 2002; hermaphroditic land snails with love-darts are a case in point. During Snook, 2005; Orr and Brennan, 2015). Examples of these courtship, a love-dart is pierced through the partner’s body wall, adaptations, in males, include increased sperm mobility to reach thereby introducing accessory mucous gland products. This mucus the sperm-storage organ faster (e.g. domestic fowl; Birkhead et al., physiologically increases paternity by inhibiting the digestion of 1999), larger testes size to transfer more sperm under strong donated sperm. The sperm, which are packaged in a spermatophore, competition (e.g. frog Crinia georgiana; Dziminski et al., 2010) are exchanged and received in an organ called the diverticulum. and transfer of accessory gland substances that influence female Because of its length, this organ was previously proposed to be a physiology (reviewed by Gillott, 2003). Common effects include female anatomical adaptation that may limit the dart interference with reduced female remating rate to enhance storage of donated sperm the recipient’s sperm usage. For reproductive success of the donor, (e.g. fruit fly Drosophila melanogaster; Chapman and Davies, an anatomically long spermatophore, relative to the partner’s 2004) and altered sperm usage by inducing oviposition following diverticulum, is beneficial as sperm can avoid digestion by exiting mating (e.g. cotton bollworm Helicoverpa armigera; Jin and Gong, through the spermatophore’s tail safely. However, the snail Eobania 2001). In response, females may physiologically resist such male vermiculata possesses a diverticulum that is three times longer than manipulations by modifying the targeted receptors or by increasing the spermatophore it receives. Here, we report that the love-dart the threshold at which male products are effective (Holland and mucus of this species contains a contraction-inducing substance that Rice, 1998; Rowe et al., 2003). This could occur in response to shortens the diverticulum, an effect that is only properly revealed sexual conflict that leads to an arms-race between male adaptations when the mucus is applied to another helicid species, Cornu and female counter-adaptations (Arnqvist and Rowe, 2002), by aspersum. This finding suggests that E. vermiculata may have which females reduce the direct costs imposed by the manipulation evolved a physiological resistance to the manipulative substance and can select for males that are able to overcome such resistance received via the love-dart by becoming insensitive to it. This provides (Cordero and Eberhard, 2003; Eberhard, 1996, 2009). However, in useful insight into the evolution of female resistance to male this context it is useful to note that female choice, in general, can manipulations, indicating that it can remain hidden if tested on a vary plastically over a reproductive season or life time (e.g. Lynch single species. et al., 2005), thus adding a layer of complexity to the interpretation. Just like in many separate-sexed organisms, sperm of different KEY WORDS: Allohormone, Antagonistic coevolution, Diverticulum, donors can also co-occur in the female reproductive system of Love-dart, Sexual conflict, Sexual selection simultaneous hermaphrodites (i.e. organisms with functioning male and female reproductive organs at the same time) as these organisms INTRODUCTION also mate promiscuously (Baur, 1998; Michiels, 1998; Koene, 2017). Males are generally in competition for reproductive access to The male function of simultaneous hermaphrodites also transfers females prior to copulation (Andersson, 1994). However, when accessory gland substances that physiologically affect sperm usage by females mate promiscuously, ejaculates of rival males can compete the mating partner (Zizzari et al., 2014). An interesting case is for egg fertilization inside the female reproductive system, referred presented by the love-dart of land snails. This accessory reproductive to as sperm competition (Parker, 1970). This form of sexual device is a calcareous stylet with a species-specific shape (reviewed selection and the potential selection on females to selectively use by Lodi and Koene, 2016b). The love-dart pierces the partner’sbody wall during courtship, in a behaviour called dart shooting (Tompa, 1984), while holding accessory mucous gland products on its surface. 1Section of Animal Ecology, Department of Ecological Science, Faculty of Earth and Life Sciences, VU University Amsterdam, De Boelelaan 1085, Amsterdam 1081 HV, In the model species the brown garden snail Cornu aspersum,the The Netherlands. 2Naturalis Biodiversity Center, Vondellaan 55, Leiden 2332 AA, mucus that enters the partner’s haemolymph has been shown to cause The Netherlands. two temporary changes in the female reproductive system (Koene and *Author for correspondence ([email protected]) Chase, 1998; for a visualization of the effects, see supplementary movie 1 in Lodi and Koene, 2016a). The first change can be measured M.L., 0000-0002-6757-0433 as waves of muscular contractions that close the entrance to the sperm- This is an Open Access article distributed under the terms of the Creative Commons Attribution digesting organ, the bursa copulatrix, and have been shown to delay License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. sperm digestion. As a result, more sperm are stored and the successful dart user can double its paternity (Chase and Blanchard, 2006). The Received 26 September 2016; Accepted 20 December 2016 second change involves the diverticulum, the spermatophore- Journal of Experimental Biology 1026 RESEARCH ARTICLE Journal of Experimental Biology (2017) 220, 1026-1031 doi:10.1242/jeb.149963 receiving organ (i.e. the organ that receives the package containing conserved the effects of the dart mucus are, so we did not test sperm). Under the influence of dart mucus, contractions of this blind- whether such female physiological responses also occurred in ended duct are initiated, possibly making spermatophore uptake easier E. vermiculata. Thus, to investigate this prediction, we here studied (Koene and Chase, 1998). the extent of the shortening effect on the diverticulum of In this scenario, the female function of dart-bearing snails may C. aspersum by performing a dose–time response test. resist the manipulation of the dart. So far, only morphological Subsequently, we assessed whether this effect is specifically co-evolutionary patterns have been shown by an inter-species caused by mucus carried on the love-dart and, importantly, comparison (Koene and Schulenburg, 2005). That study found that whether the shortening response also occurs in E. vermiculata. in species where the shape of the dart increased the surface available to transfer mucus (e.g. by blades and perpendicular blades), the MATERIALS AND METHODS diverticulum appeared and became longer. Thus, the diverticulum Adult snails of the species Cornu aspersum (O. F. Müller 1774) has been proposed to be a female anatomical adaptation to counter and Eobania vermiculata (O. F. Müller 1774) were obtained from a dart manipulation. This is also based on the finding that sperm need snail farm (Euro Helix, Cherasco, Italy). The rearing conditions to leave safely through the spermatophore’s tail when it protrudes were 20°C, a reversed photoperiod (16 h light:8 h dark) and 60% from the diverticulum entrance into the vaginal duct in order to humidity. Twice a week, the snails were cleaned, fed lettuce ad reach the site of storage (Lind, 1973), so the length of the libitum and snail feed as a source of calcium (‘Chase’ mix; see Lodi diverticulum may limit the ability of the dart to interfere with the and Koene, 2016a). Before experiments began, snails were kept process of sperm digestion. This idea is strengthened by the positive individually for at least 2 weeks on moist paper in plastic boxes correlation found between the length of the spermatophore’s tail and (11.5×11.5×5 cm) to ensure that mucus would be available in the diverticulum (Koene and Schulenburg, 2005). However, the glands for the tests. The set-up used was a portion of the resistance to the dart probably also extends to the physiological reproductive system of either species, hereafter called ‘preparation’ and biochemical level. A recent cross-reactivity test between species (for a visualization, see Lodi and Koene, 2016a), kept in a small showed that the dart mucus of Eobania vermiculata caused a dish with 2 ml saline solution at pH 7.8 (control saline; Goldstein temporary contraction of the diverticulum of Cornu