Chemical and Physical Cues Synergistically Affect Mating Behavior Sequences of Male Dasylepida ishigakiensis (Coleoptera: Scarabaeidae) Author(s): Nao Fujiwara-Tsujii, Hiroe Yasui, and Norio Arakaki Source: Zoological Science, 31(9):553-558. 2014. Published By: Zoological Society of Japan DOI: http://dx.doi.org/10.2108/zs130212 URL: http://www.bioone.org/doi/full/10.2108/zs130212

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¤ 2014 Zoological Society of Japan

ZOOLOGICAL SCIENCE 31: 553–558 (2014)

Chemical and Physical Cues Synergistically Affect Mating Behavior Sequences of Male Dasylepida ishigakiensis (Coleoptera: Scarabaeidae) Nao Fujiwara-Tsujii1*†, Hiroe Yasui1†, and Norio Arakaki2 1

Laboratory of Insect Interaction Research, National Institute of Agrobiological Sciences (NIAS), Ohwashi, Tsukuba, Ibaraki 305-0851, Japan 2 Okinawa Prefectural Agricultural Research Center (OPARC), Miyakojima Branch Office, Miyakojima, Okinawa 906-0012, Japan

We investigated physical and chemical cues involved in male mating behavior of the white grub beetle, Dasylepida ishigakiensis (Scarabaeidae). When presented with female attractant pheromone (R)-2-butanol lures in a flight tunnel, nearly all males exhibited orientation and touching behaviors to freshly killed males and females and to intact glass models. Males landed and bent their abdomens on male and female bodies, but not on intact glass models. When treated with one female equivalent (FE) extract, washed immature male bodies and glass models both evoked stronger male responses than untreated equivalents, with the former eliciting a greater response than the treated glass models. Male responses to target male and female bodies decreased with increased numbers of washings of target bodies with organic solvents. These results suggest that the chemical factors that elicit male abdominal bending behavior are present on the body surface in both sexes. Washed immature male bodies treated with 1 FE or one male equivalent (ME) of extract induced strong male abdominal bending behavior. Washed mature female bodies treated with 1 ME extract also evoked male responses. Extracts of both sexes included factors eliciting male abdominal bending behavior. These results suggest that both physical and chemical cues derived from conspecifics cooperate to facilitate male mating recognition in D. ishigakiensis. The mating process of this species in the field is highly synchronized. Thus, after orienting to a female-like object, the only information males require by touching is whether the sex attractant pheromone that attracted them is indeed from a conspecific. Key words: abdominal bending, contact pheromone, flight tunnel, male mating behavior, Dasylepida ishigakiensis INTRODUCTION The white grub beetle, Dasylepida ishigakiensis Niijima et Kinoshita (Coleoptera: Scarabaeidae), is one of the most destructive sugarcane pests in the Miyako Islands, Okinawa Prefecture, Japan (Sadoyama et al., 2001). To develop methods to control this insect, studies have been conducted on its life cycle (Oyafuso et al., 2002; Arakaki et al., 2004; Tanaka et al., 2008) and pheromonal communications system, the sex attractant pheromones in particular (Wakamura et al., 2009a, b). The nonvolatile surface chemical features of D. ishigakiensis have only been investigated in a single study by Hirai et al. (2008). In our previous flight tunnel experiments on the white grub beetle, males oriented to female attractant pheromone lures comprising (R)-2-butanol (R2B), but did not bend their abdomens toward visual models made from brown cotton * Corresponding author. Tel. : +81-29-838-6205; Fax : +81-29-838-6205; E-mail : [email protected] † These authors contributed equally to this article. doi:10.2108/zs130212

balls (Wakamura et al., 2009a; Fig. 1A). Under the same conditions, however, males sometimes bent their abdomens toward glass models treated with female crude extract and immediately did so towards dead females (Fig. 1B). Based on these observations, we hypothesize that white grub beetles possess contact pheromones on their bodies that evoke copulation behavior in males. Many studies have demonstrated that beetles, particu-

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Fig. 1. Male behavior in the flight tunnel in response to models tied to lures of the female sex attractant pheromone (R)-2-butanol. (A) Orientation behavior to a brown cotton ball. (B) Abdominal bending behavior to a freshly killed female.

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larly the Cerambycidae (Fukaya et al., 1996; Crook et al., 2004; Lacey et al., 2008; Yasui et al., 2009; Silk et al., 2011), use contact sex pheromones in mating (Silk et al., 2009). Both sex attractants and contact pheromones are reported to play important roles for mating success in longhorn beetles (Cerambycidae) (Allison et al., 2004). There are few reports concerning contact pheromones in Scarabaeidae (Fombong et al., 2012), so a complete understanding of the mating sequence of D. ishigakiensis will expand our knowledge of the beetle’s ecology. In this paper, we characterize the physical and chemical cues involved in D. ishigakiensis mating behavior to gain a better understanding of their communication system. MATERIALS AND METHODS Insects Third-instar larvae of D. ishigakiensis were collected from the soil in sugarcane fields on Miyako Island in early February 2012. The larvae were individually kept at 24°C on a long photoperiod (14L:10D) and maintained in plastic cups (inner diameter, ca. 5.7 cm; height, 3.5 cm), each containing humus and fertile soil as substrate and a piece of sugarcane stem (diameter, ca. 1.5 cm; length, 2 cm) as food. The sugarcane stems were changed every three weeks until the larvae stopped feeding. The larvae pupated 6–8 months after collection, and the pupae reached adulthood approximately four weeks thereafter. Adults terminate reproductive diapause and become sexually mature if they are first kept at 25°C for one week and then exposed to 20°C or 15°C for eight weeks (Tanaka et al., 2008). Feral males for experiment 3 of the flight tunnel assay were also collected by visual searching or with R2B-baited traps in sugarcane fields in early February 2014. Those males were brought to the NIAS laboratory in Tsukuba, Japan. They were individually kept with moist absorbent wiper (JK wiper®, Nippon Paper Crecia, Tokyo, Japan) in plastic cups at 16°C until use. Chemicals R2B (> 99% purity; > 98% ee) was purchased from Wako Pure Chemical Industries (Osaka, Japan). The lures used in the flight tunnel bioassay were prepared in the laboratory of the Shin-Etsu Chemical Company (Joetsu, Japan). R2B was sealed in Type A high-density polyethylene tubes, as described by Wakamura et al. (2009b). The inner and outer diameters of the tube were 0.84 mm and 1.54 mm, respectively. The Type A tubes were 1 cm long and contained approximately 5.0 mg of the R2B. The release rate of the pheromone was estimated at approximately 3.5 ng per min (100 ng per 30 min) at approximately 23°C in the laboratory using the method established by Yasui et al. (2010). The amount of R2B emitted from this lure over 30 min corresponded to the total 2-butanol emission of a single female during one instance of calling (Yasui et al., 2010; Wakamura et al., 2009a). Flight tunnel assay Behavioral assays were performed using the flight tunnel that was developed by Yasuda (1996) to evaluate male mating behavior towards female models. The assay followed the basic protocol described in previous research (Fukaya et al., 2009; Wakamura et al., 2009a). The flight tunnel was made of transparent acrylic plates and was 75 cm long, 18.5 cm wide, and 18.5 cm high. A fine wire screen separated the tunnel’s flight and windward chambers, and a second wire screen was affixed to the tunnel’s other end. An electric fan supplied air inside the flight tunnel at approximately 20 cm/s, and outlet air was exhausted to the outside of the tunnel. The main tunnel floor was covered with a sheet of white paper. A sealed R2B lure was attached to various models using thin

wire and placed at the center of the flight tunnel 10 cm from the tunnel’s windward end. One male was placed on a paper disk (9 cm in diameter; No. 2 filter paper, Toyo Roshi Kaisha, Tokyo, Japan) in a plastic container (5 cm high) at the downwind end of the flight tunnel. The male was initially covered with a transparent plastic cup (6 cm diam. × 4 cm height), which was opened to expose it to R2B once observations began. Males were 59–63-day-old virgins, and were used in the assay once per day. Individual males were used a maximum of four times. Data on males that could not fly are omitted. The flight tunnel floor was lit by a 40 W light bulb and light intensity was decreased gradually from 175 to 0.91 lx. This change in light intensity is roughly equal to that encountered when mating behavior has been observed in sugarcane fields (Arakaki et al., 2004; Fukaya et al., 2009). Under these conditions, on taking flight, the male shows the following sequence of behaviors towards a positive female model: hovers within 5 cm of the leeside of the female model (orientation), touches it several times, lands on it, bends his abdomen, and attempts to copulate. Of these behaviors, touching, landing, and abdominal bending were recorded. Each assay was continued until the male stopped flying (21 min maximum). The experiments were conducted 1.5 h before the light was turned off (at approximately dusk of their controlled light cycle) in the laboratory at approximately 22°C. The number of males used in each test sample is shown in the figures. Experiment 1. Male responses to freshly killed males and females and to glass models Male responses to freshly killed mature and immature males and females and to glass models were recorded. All individuals were killed by freezing (−30°C). Models were brown glass molded into beetle shapes (15 × 9 × 6 mm). To prevent the males from slipping and falling, all smooth surfaces of the model were filed. Male responses, such as touching, landed on, and abdominal bending toward the models, were observed. Experiment 2. Male responses to intact and solvent-washed adult bodies This species has an adult diapause that serves to synchronize the time of sexual maturation; males cannot copulate with immature females that have not experienced adult diapause (Tanaka et al., 2008). In this experiment, we observed male responses to mature adults (59d) that had experienced adult diapause, and immature adults (7d), which had recently eclosed. Mature and immature males and females were killed by freezing (−30°C). Solvent-washed bodies were obtained by soaking some individuals in n-hexane and ethyl acetate. Those two solvents possess different polarities, allowing us to remove a wide range of chemicals from the body surfaces. Twice-extracted individuals were soaked once for 10 min in both solvents (wash × 2), four-times extracted individuals were soaked twice for 10 min in both solvents (wash × 4), and eight-times extracted individuals were soaked four times for 10 min in both solvents (wash × 8). Male abdominal bending responses to each model were recorded. Experiment 3. Male responses to washed adult bodies treated with female or male extract To examine the differences between pheromonal activities of female and male body extracts, and to compare the physical stimuli of male and female bodies, we conducted replacement experiments. To obtain female and male extracts, mature beetles were individually soaked in n-hexane (1 ml per individual, female equivalent = FE, male equivalent = ME) for 10 min. These crude extracts were concentrated and 10 μl were applied to wash × 2 immature males and wash × 8 mature females. Male abdominal bending responses to each model were recorded. Feral males were used for this experiment.

Male mating behavior of D. ishigakiensis Experiment 4. Male responses to glass models and washed immature male bodies treated with female extract The crude female extract was concentrated and applied to glass models and to washed immature males (1FE / 10 μl). To test the effects of chemical stimuli derived from the female extract, we used washed immature male bodies as a substrate; wash × 2 immature male bodies were the only model that had activity equally low to the untreated glass model. Male abdominal bending responses to those models (intact and 1 FE treated washed immature male body and glass model) were recorded. Experiment 5. Dose response Immature wash × 2 male bodies were treated with varying doses of female extract (2, 1, 0.5, 0.2, 0.1, 0.01, and 0.001 FE as a 10-μl hexane solution). Male abdominal bending responses to each test model were recorded.

ing increased (90%, 48%, and 23% for intact, wash × 2, and wash × 4, respectively) (Fig. 3B). There was no significant difference between the activities of immature and mature female bodies (90% and 100%, respectively), but intact immature male bodies were statistically less attractive than mature male bodies (53% and 90%, respectively) (Fig. 3). Experiment 3 When washed immature male bodies were treated with male and female extracts, male abdominal bending responses were significantly higher than those of untreated or wash × 2 male bodies (65%, 54% and 23%, respectively) (Fig. 4). No significant difference was observed in the effects of female and male extracts. Similarly, male responses to washed male and female bodies treated with male extract

Statistics To compare rate values obtained in the flight tunnel assay, if the n × 2 chi-squared test was significant (P < 0.05), the paired chisquared tests were subsequently calculated. Significance was determined with Bonferroni’s corrected P value (Sokal and Rohlf, 1995).

Experiment 1 All males used in bioassays showed orientation and touching behavior to freshly killed males and females and to intact glass models (Fig. 2). However, behaviors such as landing and abdominal bending were rarely observed towards glass models. All males landed on both males and females, while only 40% of males landed on glass models. Similarly, 100% and 90% of males showed abdominal bending behavior to female and male bodies, respectively, but only 5% of males showed that behavior to glass models. Experiment 2 Wash × 4 mature female bodies were as attractive to males as intact female bodies (77%) (Fig. 3A). However, male responses to washed male bodies decreased as wash-

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were both 54%, significantly higher than that to washed male bodies.

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Fig. 5. Male abdominal bending responses to glass models and immature male bodies treated with one female equivalent of mature female extract. The vertical axis represents the number of males responding per number taking off. Values accompanied by the same letter are not significantly different at the 5% level. The numbers in parentheses are the numbers of males examined (= no. taking off).

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Experiment 4 A significant effect was found when female extract was applied to glass models and wash × 2 immature male bodies (Fig. 5). Glass models treated with 1 FE of extract elicited higher male responses than intact glass models, although abdominal bending behavior was observed at a low level (20%). In contrast, when the same amount of female extract was applied to washed male bodies, male responses were much higher (80%).

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Experiment 5 Male bodies treated with 2 to 0.1 FE elicited a high response rate and were not significantly different among treatments (71%, 80%, 57%, 60%, and 60%, respectively). However, the dose of female extract had a significant effect on male responsiveness to models among the 0.1 to 0.001 FE treatments (60%, 38%, and 25%, respectively) (Fig. 6). Males treated with 0.001 FE were still statistically more attractive than wash × 2 immature male bodies.

Fig. 6. Male abdominal bending responses to twice-washed, immature male bodies treated with different doses of mature female extract. Untreated, twicewashed immature bodies were used as control. The vertical axis represents the number of males responding per number taking off. Values accompanied by the same letter are not significantly different at the 5% level. The numbers in parentheses are the numbers of males examined (= no. taking off).

DISCUSSION This study examined the effects of contact pheromones in eliciting male abdominal bending behavior in D. ishigakiensis. In addition to chemical factors, we demonstrated the synergistic effects of physical cues in eliciting this behavior. Although intact glass models did not induce males to bend their abdomens, freshly killed male and female bodies equally evoked this behavior (Fig. 2). This suggests that both male and female bodies are equally attractive to males. Observations from both field and laboratory experiments (Fujiwara-Tsujii, personal observation) have shown that

males attempt to copulate with both males and females. These observations are consistent with the results in Fig. 2. As the number of washes with organic solvents increased, male responses to washed male bodies decreased (Fig. 3). In the case of mature females, wash × 8 bodies evoked significantly low levels of male abdominal bending behavior. Furthermore, sexually mature males elicited stronger responses than immature individuals, and immature female bodies lost activity with only wash × 2 treatments. Solvent extractable, chemical cues eliciting abdominal bending behavior may increase during sexual development. Replacement experiments were conducted to compare

Male mating behavior of D. ishigakiensis

the pheromonal activities of female and male body extracts, and to test the difference between the physical stimuli of bodies of the two sexes (Fig. 4). Male and female extracts equally increased male responsiveness when applied to washed immature male bodies. Furthermore, washed female bodies treated with male extract also elicited the same levels of male response. These results suggest that contact pheromones are present in the extracts of both sexes. Because males responded equally to the physical stimuli of male and female bodies in this experiment, we washed and used immature male bodies as substrates to assess chemical cues. When treated with 1 FE extract, glass models evoked higher male responses than untreated glass models. This result suggested that chemical stimuli derived from female extracts evoked male responses. As stated above, wash × 2 immature male bodies did not evoke male abdominal bending behavior. Washed immature male bodies treated with 1 FE extract elicited a higher response than similarly treated glass models. Male responses were highest when physical cues of the immature male body worked synergistically with chemical factors of the female extract (Fig. 5). In addition to the results of Fig. 4, these results suggest that both physical and chemical cues derived from conspecific bodies work together to facilitate male mating recognition in D. ishigakiensis. When both chemical and physical factors were present, 0.001 FE extract-treated samples still elicited statistically higher activity than the control (washed immature males) that represented only physical cues (Fig. 6). This result demonstrated that males can detect and react to the contact pheromone even at a very low concentration. Based on the results of this study and our previous work, the assumed mating sequence of D. ishigakiensis is as follows. First, a gradual decrease in ambient light induces male mating flight (Fukaya et al., 2009). Second, after takeoff, a male detects the female sex attractant pheromone, R2B, and orients to a female-like object near the odor (Wakamura et al., 2009a). Throughout our flight tunnel bioassay, when R2B was not present, males did not show orientation behavior even after take-off (Wakamura et al., 2009a), suggesting that male mating behavior is initiated by R2B and will not occur without exposure to this pheromone. Females that can emit detectable amounts of R2B are those that are reproductively mature (Fujiwara-Tsujii et al., 2011) and ready to copulate. Third, after approaching and touching the female-like object, males will land on it if they detect the conspecific contact pheromone. When both chemical and physical cues are detected, males will bend their abdomens for copulation. Males can find females by their sex attractant pheromone, and so after approaching the object near the R2B scent, the last cue for eliciting landing and abdominal bending behavior is whether the object is the same species or not. However, we have not compared the chemical profiles of the surface chemicals of the two sexes. Chemical analytical investigations should be conducted in the future. The mating systems of Cerambycidae have been the most extensively studied of any beetle. Their mating sequences and female specific long- or short-range attractant pheromones are well reported (Allison et al., 2004). However, the mating systems of cerambycid beetles are

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quite different from those of some of Scarabaeidae, including D. ishigakiensis. For example, the durations of mating behavior are limited, and males do not fly long distance to find mates (Arakaki et al., 2004; Kawasaki and Tamaki, 1985). Their behavioral ecology may cause their lack of female-specific contact pheromones. Male-to-male copulation attempts were observed in other Scarabaeidae may be due to the absence of a female-specific contact pheromone (Leal, 1991; Leal et al., 1997). Dasylepida ishigakiensis has two diapauses that appear to synchronize the beetle’s long life cycle with the seasons (Tanaka et al., 2008). By using the cues of light intensity and temperature at 18:00, they synchronize the timing of mating flights (Oyafuso et al., 2002; Arakaki et al., 2004; Fukaya et al., 2009). Under such highly-synchronized mating-flight situations, the only information males require after orienting to a female-like object is whether the chemical signal is indeed from a conspecific. The highly-synchronized mating mechanisms of this beetle may be enable males to successfully copulate without the female-specific contact pheromone reported in other species (Rutledge et al., 2009; Silk et al., 2009). ACKNOWLEDGMENTS This work was supported by research and development projects for the application and promotion of new policies for agriculture, forestry, and fisheries (No. 23021). We thank Hiroaki Oroku, former director of the Miyakojima branch of OPARC, for his kind support of our project. Fumiaki Mochizuki of the Shin-Etsu Chemical Co. kindly prepared the R2B lures for the flight-tunnel bioassays. We also wish to thank Seiji Tanaka of NIAS and Atsushi Nagayama of OPARC for their cooperation and stimulating discussions. Thanks are also due to Yuko Shimizu, of the Okinawa Prefectural Plant Protection Center, for her cooperation in collecting larvae, and to Ikuko Hashimoto, who helped rear the beetles at NIAS and assisted us in our experiments.

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Chemical and Physical Cues Synergistically Affect Mating Behavior Sequences of Male Dasylepida ishigakiensis (Coleoptera: Scarabaeidae).

We investigated physical and chemical cues involved in male mating behavior of the white grub beetle, Dasylepida ishigakiensis (Scarabaeidae). When pr...
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