DOI: 10.1111/eea.12740 The mating systems of three species of minute pirate bug, Orius sauteri, O. minutus,andO. strigicollis Toru Arakawa, Kiyoko Taniai & Taro Maeda* Insect Interaction Research Unit, National Institute of Agrobiological Sciences, NARO, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan Accepted: 13 August 2018 Key words: polygamous, monandrous, natural enemies, behaviour, morphology, genitalia, SEM, biocontrol agents, biological control, polygamy, Heteroptera, Anthocoridae Abstract We compared the mating systems and genital morphology of three Orius species dominant in Japan: Orius sauteri (Poppius), Orius minutus (L.), and Orius strigicollis (Poppius) (Heteroptera: Anthocori- dae). In all three species, the males were polygamous and could inseminate at least three females at a rate of one female per day. Compared to the other two species, the O. minutus males had a lower rate of success in three consecutive mating orders. The mated females laid the same number of eggs irre- spective of mating sequence. Orius minutus and O. strigicollis females were found to be monandrous, and they re-mated with another male only when the 1st mating failed. In contrast, O. sauteri females were polyandrous and accepted a 2nd male even when the 1st mating was successful. Multiple mating with a single male did not increase their fecundity, and it decreased the hatching success of eggs. Mat- ing with multiple males did not affect the fecundity or hatching success. Our scanning electron microscopy observation suggested that the genitalia of O. minutus and O. strigicollis were more simi- lar in shape. The similarities in mating system and morphology between O. minutus and O. strigicollis support a close phylogenetic relationship. polyandry (Fisher et al., 2013), in which ‘sexy sperm’ will Introduction be inherited (Yasui, 1998). Under different conditions, Most studies of mating systems of insect natural enemies polyandry could be chosen. If it is costly to reject sexual have been conducted to understand either the evolution of harassment by males, females accept males’ attempts; this the mating systems themselves or the evolution of sex allo- is called convenience polyandry (Arnqvist & Nilsson, cation decisions (Hardy et al., 2005). The evolutionary 2000; Rowe & Arnqvist, 2002). consequences of polyandry are of interest, as it is not yet Minute pirate bugs (which make up the genus Orius) established why females would mate with multiple males are effective predators of important agricultural pests (Pizzari & Wedell, 2013). Mating involves various costs, including thrips, spider mites, aphids, and whiteflies (Lat- such as time and energy, predation risk, physical injury, tin, 1999). Many studies of Orius species have focused on and parasite/pathogen infection (Arnqvist & Nilsson, their foraging and reproductive ability because of their 2000). If the benefit of re-mating exceeds these costs, importance in biological control. In contrast, there have females should favour polyandry (Arnqvist & Nilsson, been only a few studies on the mating system of Orius. 2000; Arnqvist & Andres, 2006). Non-genetic benefits of Leon-Beck & Coll (2009) revealed that Orius laevigatus re-mating, among other benefits, are adequate sperm (Fieber) females are monandrous and males of the species (Elzinga et al., 2011), direct benefit from ejaculate compo- are polygamous. In contrast, Kobayashi & Osakabe (2009) nents (Paukku & Kotiaho, 2005; Boulton & Shuker, 2013), demonstrated that Orius sauteri (Poppius), Orius strigicol- nuptial gifts (Gwynne, 2008), and fertilization success. lis (Poppius), and Orius nagaii Yasunaga females displayed Genetic benefits are mating with a ‘good’ male and getting multiple mating with several males. If the genus Orius ‘good’ genes (Tregenza & Wedell, 1998, 2000; Kuijper includes both polyandrous and monandrous species, it is et al., 2012). Sperm competition could occur in true worthwhile to compare the mating systems of closely related species in order to help clarify the evolution of *Correspondence: E-mail: [email protected] polyandry. © 2018 The Netherlands Entomological Society Entomologia Experimentalis et Applicata 167: 141–151, 2019 141 142 Arakawa et al. Horton & Lewis (2011) suggested that Orius species O. minutus demonstrate traumatic insemination in which males use external genitalia to pierce and penetrate the female abdominal wall and ejaculate into the abdominal cavity during copulation. Although females’ defensive mecha- nisms have evolved, the mating costs in species with trau- O. strigicollis matic insemination are slightly higher compared to those of other species (Reinhardt et al., 2014). Indeed, physical damage can reduce longevity and reproductive success (Stutt & Siva-Jothy, 2001; Morrow & Arnqvist, 2003; O. sauteri Backhouse et al., 2012; Tatarnic & Cassis, 2013; Reinhardt et al., 2014). In discussions of the mating system of Orius spp., sexual conflict caused by traumatic insemination should be considered. On mainland Japan, O. sauteri, Orius minutus (L.), O. O. nagaii strigicollis,andO. nagaii are commonly distributed in a Figure 1 The phylogenetic relationships of four Orius species that wide area across Japan (Yasunaga, 1997a,b,c). These spe- are commonly distributed in a wide area across Japan. cies often occur sympatrically on various flowering plants (Ohno & Takemoto, 1997; Hinomoto et al., 2009) and have similar prey, including thrips, aphids, and hatchlings females in the 3rd experiment to determine whether the and larvae of various insects. Three of these species – O. females are monandrous or polyandrous. Notably, even if sauteri, O. minutus,andO. strigicollis – display good females show multiple mating with multiple males, it does potential as biological control agents (Ohno & Takemoto, not always mean they are polyandrous. If subsequent cop- 1997). A previous investigation of the distribution of these ulation occurs only when the previous copulation failed, three indigenous species in Japan revealed that two or all this is considered a form of monandry and is referred to as three species often coexisted on the same plants (Hino- ‘pseudopolyandry’ (Fisher et al., 2013). To distinguish moto et al., 2009). In addition to their similar habitat, the pseudo- from true polyandry, we checked whether the 1st three species’ prey preference, body size, and appearance mating was successful. We conducted the 4th experiment are very similar. to determine whether mating has non-genetic benefits. As The authors of a phylogenetic study suggested that O. multiple mating with a single male does not relate to sauteri, O. minutus,andO. strigicollis are closely related, genetic benefits, we allowed females to mate multiple times and that the relationship between O. minutus and O. strigi- with single males. In the 5th experiment, we compared the collis is closer than that between either species and O. sau- fecundity and hatching success of females mated with a teri (Muraji et al., 2000a,b; Figure 1). Morphological single male or three males. We discussed the genetic bene- observations of these three species (Yasunaga, 1997b) fits of polyandry based on the results of experiments 4 and show good agreement with the phylogenetic trees (Muraji 5. For the 6th experiment, we conducted a detailed scan- et al., 2000a). We were intrigued by the question whether ning electron microscopy (SEM) comparison of the male the mating systems of these Orius species would also coin- genitalia and mating systems of O. sauteri, O. minutus,and cide with their phylogenetic and morphological traits. It is O. strigicollis. known that genital structures vary widely in the family Anthocoridae, and we speculated that differences in genital Materials and methods structures among O. sauteri, O. minutus,andO. strigicollis might be involved in the differences in their mating beha- Insects viours and mating systems. Orius sauteri and O. minutus were collected from red clo- We conducted six experiments with the three species. In ver and chestnut, respectively, in Ibaraki, Japan. Orius the 1st experiment, we observed the species’ mating beha- strigicollis was obtained from a commercial source (Arysta viour and recorded the duration of copula and the number Life Science, Tokyo, Japan). The insects were maintained of eggs to observe what constituted effective copulation for in a plastic case (24 9 17 9 5cm)usingEphestia kueh- fertilization. In the 2nd experiment, we focused on the niella Zeller eggs (Ga-Ran; Agrisect, Inashiki, Ibaraki, mating capability of the males; we counted how many Japan) as a diet source and Sedum mexicanum Britt. twigs females a male could inseminate and how many eggs were as oviposition substrates under 25 Æ 1 °C, 60–70% r.h., deposited. We investigated mating frequency of the and a L13:D11 photocycle. A mesh sheet (15 9 10 cm) Mating system of three minute pirate bugs 143 was put on the bottom of the plastic case as a shelter for eggs deposited was assessed. Unlike most Heteroptera, the immature bugs. A plastic tube (1 cm diameter, 7.5 cm Anthocoridae (to which the Orius species belong) have a long) filled with water and plugged with cotton wool was unique fertilization system, in which fertilization takes provided as a moisture source. To obtain similarly aged place before the chorion is deposited (Horton, 2008). adult virgin bugs for the experiments, 4th or 5th instars Oocyte maturation does not occur in unmated females. were picked up from the plastic case and isolated individu- Unfertilized eggs generally degenerate and are resorbed