Research Article Journal of Orthoptera Research 2024, 33(2): 249-254 Mating behavior and three types of mating songs of the sandy beach- dwelling ground cricket Dianemobius csikii (Grylloidea, Trigonidiidae, Nemobiinae) TAKASHI KURIWADA! 1 Laboratory of Zoology, Faculty of Education, Kagoshima University, Korimoto 1-20-6, Kagoshima 890-0065, Japan. Corresponding author: Takashi Kuriwada (kuriwada@edu.kagoshima-u.ac.jp) Academic editor: Ming Kai Tan | Received 23 June 2023 | Accepted 19 February 2024 | Published 16 August 2024 https://zoobank. org/33757297-D588-4333-B855-1CCDE40A4029 Citation: Kuriwada T (2024) Mating behavior and three types of mating songs of the sandy beach-dwelling ground cricket Dianemobius csikii (Grylloidea, Trigonidiidae, Nemobiinae). Journal of Orthoptera Research 33(2): 249-254. https://doi.org/10.3897/jor.33.108536 Abstract This study aimed to investigate the mating behavior of the sandy beach-dwelling ground cricket, Dianemobius csikii (Bolivar, 1901) (Orthop- tera, Grylloidea, Trigonidiidae, Nemobiinae). The calling songs of males were recorded, and the temporal structures of the songs were analyzed. Subsequently, the courtship song and mating behavior of cricket pairs were observed. The calling song consisted only of monotonous chirps, while the courtship song consisted of similar chirps and ticks consisting of a single pulse. Dianemobius csikii exhibited a relatively longer courtship duration than other cricket species. The female stayed with the male for approxi- mately 25 min while the male emitted the courtship song. The male then changed to the trill song, which is a continuous song, just before copula- tion. Copulation occurred within 10-40 s of the male emitting the trill song. The courtship behavior differs from that of other well-studied cricket species, such as Gryllinae. The findings of this study provide insight into the mating behaviors of crickets. Keywords calling song, courtship song, Orthoptera, sexual selection, Trigonidiidae Introduction In many animal species, males produce signals for mating (Andersson 1994). These males often produce different types of signals at different stages of reproduction (e.g., nuptial colora- tion and behavioral display in the guppy Poecilia reticulata, Houde 1997; some mating call types in frogs and toads, Kelley 2004). Understanding how and why multiple signals occur is currently a key topic in sexual selection research (Hebets and Papaj 2005, Bro-Jorgensen 2010, Partan 2013, Heinen-Kay et al. 2021). For ex- ample, different signals provide different information to receivers, including redundant backup information, distinct and comple- mentary information, or signal amplification (Candolin 2003). In crickets for several well-studied species, especially for Gryl- linae, males produce three types of acoustic signals: calling, court- ship, and aggressive songs (Alexander 1961, Gerhardt and Huber 2002). Calling songs are used for the long-range attraction of fe- males and the demonstration of territory to other males, court- ship songs are used in short-range courtship immediately prior to copulation, and aggressive songs are produced during fighting behavior with rival males (Alexander 1961, Gerhardt and Huber 2002). These song characteristics play a crucial role in the repro- ductive success of males. The bulk of research on the acoustic communication of crick- ets has focused on Gryllinae, with few studies other cricket groups. However, several species of Trigonidiidae, which includes more than 700 species and inhabits diverse environments (Orthoptero- logical Society of Japan 2006), have been reported to exhibit pe- culiar mating behavior and mating songs. For example, the sword- tail cricket Cranistus colliurides Stal, 1861 (Orthoptera: Trigoni- diidae) produces four distinct types of songs: calling, courtship, agonistic, and post-copulatory (Centeno and Zefa 2019, Centeno et al. 2021). Post-copulatory songs are rarely reported in crickets, and the function of the song is not known (Centeno et al. 2021). The striped ground cricket Allonemobius socius (Scudder, 1877) (Orthoptera: Trigonidiidae) exhibits courtship behavior depend- ing on social environments: the males often omit the courtship song and copulate with females in solitary environments (Sadows- ki et al. 2002). Similarly, A. socius exhibits complex courtship be- havior, including three types of courtship songs and two types of nuptial gifts (Mays 1971, Sadowski et al. 2002). Thus, the unique and diverse mating behaviors of Trigonidiidae make these species a valuable target for research on acoustic communication The sandy beach-dwelling ground cricket Dianemobius csikii (Bolivar, 1901) is mainly distributed along the sandy beaches of Japan, Korea, and China (Orthopterological Society of Japan 2006, 2016). It is likely that the sandy beach environment has led to various adaptations in this species. For example, the body color of the cricket closely resembles the sand of its habitat (Fig. 1; Sato and Kuriwada 2022). Unfortunately, this species is feared to be extinct in some areas of Japan due to the disappearance of sandy beaches caused by coastal development (Fukuoka Prefec- ture 2014, Kyoto Prefecture 2015). Although mating behavior in Copyright Takashi Kuriwada. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. JOURNAL OF ORTHOPTERA IRESEARCH 2024, 33(2) 250 other species of the same genus have been investigated (Kuriwada 2023), the ecology and behavior of D. csikii have not been widely studied; thus, the objective of this study was to describe the mat- ing behavior of this cricket. This study measured the temporal structures of the calling and courtship songs of D. csikii and examined the relationship between the structures of the courtship song and the copulation success of males. Furthermore, the male of D. csikii was observed emitting another type of song—a trill song—just before copulation that dif- fered from their calling and courtship songs. The details of these results of the analyses of the songs along with observations of mat- ing behavior of the cricket are reported here. Materials and methods Insect collection and rearing—Nymphs of D. csikii were collected from a sandy beach in Minamisatsuma city, Kagoshima Prefecture, Japan (31.4413°N, 130.2832°E), between late July and early Au- gust 2020. A total of 58 crickets were reared in a container (42 x 24 x 25 cm) at 27 + 1°C under photoperiods of 15 h of light to 9 h of darkness (light cycle: 04:00 to 19:00). The crickets were given egg cartons for shelter, soil in a 200 mL plastic cup, guinea pig food (Marmot selection; Yeaster Co., Ltd., Hyogo, Japan), and cat food (Purina One Metabolic Energy Control; Purina, Kobe, Japan) provided ad libitum. Furthermore, the soil was sprinkled with water every 2-3 days to provide the crickets with a source of water and a site suitable for oviposition. The individuals used in this experiment were the third-generation progeny of the crickets originally collected from the beach. The final instar nymphs were reared individually in plastic containers (diameter = 6.0 cm; height = 3.0 cm), and the cups were checked daily for newly emerged adults. Adult crickets were kept in identical individual cups until the experiment began. Recording of the calling song.—Using an uncompressed digital IC recorder with a 44.1 kHz sampling rate and a 16-bit dynamic range, the male calling song was recorded one to two days be- fore the observation of mating behavior (RR-XS455; Panasonic, Osaka, Japan). The recordings were saved as WAV files. Each male container (6 cm diameter, 3 cm high) was placed with the IC re- corder in a two-ply corrugated fiberboard box (inner side: 23 x 17 x 17 cm, outer side: 28 x 23 x 23 cm) that was physically, visually, and acoustically isolated from other cricket containers. All record- ings were made at temperatures of 27 + 1°C from 19:00 to 04:00, which corresponds to the dark period. Using Audacity v2.0.5 soft- ware (Dominic Mazzoni, Mountain View, CA, USA), seven com- ponents of the calling song were analyzed: time spent calling at night, chirp duration, chirp interval length, dominant frequency of the chirp, pulse duration, pulse interval length, and the number of pulses per chirp (Fig. 1A, D). Because pulse characteristics are important for species recognition in other cricket species (Gryl- lus bimaculatus De Geer, 1773, Sch6neich et al. 2015), pulse char- acteristics were measured in addition to chirp characteristics. For analyses of chirp components, 10 measurements per component were randomly chosen. For analyses of pulse components, five measurements per component were randomly chosen. The mean of the chosen measurements was taken as the component value for the individual. Observation of mating behavior—Unmated males and females 8-14 d after their final molt were used for the observations. A focal male was placed in a plastic container (diameter = 12 cm, T. KURIWADA A Fig. 1. A. Male Dianemobius csikii; B. Four D. csikii in the sand of their habitat; individuals indicated by black lines. height = 19.0 cm), an unmated female was added to the container after 1 min, and the mating behavior of the pair was observed for 40 min. This process was repeated until 40 pairs had been ob- served . All experiments were started between 16:00 and 18:20 un- der fluorescent light. The timing fluctuated between 40 min and 3 h before the dark cycle, when crickets are usually active (Shimizu and Masaki 1997). Mating behavior was recorded using a video camera (SONY HDR-CX270V; Sony, Tokyo, Japan) held directly above the container at a distance of 50 cm. Latency to courtship was measured from the start of cohabitation to the production of a courtship song. During courtship, the male crickets switched from emitting the courtship song (Fig. 1B) to emitting the trill song, which has a different temporal structure (Fig. 1C). The trill song was defined as a song that was not interrupted by an interval from the time it was produced to the time of copulation. Court- ship duration was defined as the time from the beginning of the courtship song to the start of the trill song. The courtship songs were recorded for 1 min using the same equipment described for the recording of the calling song. Latency to copulation was meas- ured from the start of the trill song to copulation. Almost all of the males who copulated produced the trill song. I recorded the trill song in nine males. Copulation was defined as the contact of male genitalia with female genitalia during mounting behavior. Copulation was defined as successful when a spermatophore was attached to the female genitalia. If the pair did not initiate copula- tion after 40 min, it was recorded that the male had not copulated. The recorded courtship and trill songs were analyzed similarly to the calling song. The courtship song contained two types of song components (see Results section), and thus the number of repeti- tions of the same component type was recorded. Statistical analyses. —To examine the effect of the temporal struc- ture of courtship songs on copulation success, a generalized lin- ear model with binomial error and logit link was implemented using R 4.1.2 (R Core Team 2021). The explanatory variables in this model were the components of the courtship song, and the response variable was copulation success (success = 1, failure = 0). The statistical significance of each coefficient was tested using the likelihood ratio test. Kendall’s rank correlation was used to exam- ine the correlation in each parameter between calling and court- ship songs, and paired t-tests were used to compare the temporal structures of the calling and courtship songs. Results Temporal structure of the three song types.—The median time spent calling at night was 5.40 min (25-75% quantiles: 0-125.8 min, JOURNAL OF ORTHOPTERA RESEARCH 2024, 33(2) T. KURIWADA N = 40). The calling song of D. csikii comprised only chirps (Fig. 2), whereas the courtship song consisted of a series of chirps interspersed with several short ticks (Fig. 3). The chirp consisted of approximately 65 pulses, while the tick consisted of one pulse. The pulse is the sound produced by a single rubbing of the fore- wings. The trill song maintained a continuous structure without interruption until copulation (Fig. 4). The pulse of the trill song was unclear (Fig. 4C) and could not be measured. The descrip- tive statistics for the three song types are listed in Table 1. The temporal structure of the courtship song did not affect copula- tion success (Table 2). There were no significant correlations be- tween calling and courtship songs (chirp duration: t = 0.25, p = 0.078; interval length: t = 0.13, p = 0.35; dominant frequency: tT = 0.21, p = 0.15; pulse duration: t = 0.025, p = 0.87; pulse inter- val: t = -0.20, p = 0.20; and No. pulse/chirp: t = 0.016, p = 0.91). The interval length of the courtship song was significantly shorter than that of the calling song (paired t-test: t,, = 12.38, p < 0.001), although the chirp duration of the courtship song was not signifi- cantly different from that of the calling song (t,, = 0.47, p = 0.64). The dominant frequency of the courtship song was significantly lower than that of the calling song (t,, = 2.19, p = 0.038). There were no significant differences in pulse duration or the number of pulses per chirp between calling and courtship songs (pulse Ae * 20 .- ‘NS 154 * a 20 =a a 10 Frequency (kHz) courtship song rar ow Amplitude 7) Amplitude Time (s) Fig. 4. Sound spectrogram (A) and oscillograms (B and C) of trill song of Dianemobius csikii. The oscillogram in C is an expansion of the time axis in B. Once the courtship song changed to the trill song, the trill song was continuously emitted until copulation began. Dotted lines B to C show the approximate corresponding times. JOURNAL OF ORTHOPTERA RESEARCH 2024, 33(2) T. KURIWADA A. Approach B. Courtship song | {y mR * C. Trill song 2.9 % D. Mounting E. Disengage Fig. 5. Mating behavior of Dianemobius csikii. A. Male emitting the calling song. B. When a part of the female body touches that of the male, the male emits the courtship song. The fe- male stays by the male during the courtship song. All males (40 individuals) emitted a courtship song when approached by a female. C. Approximately 25 min after the male begins to pro- duce the courtship song, most males (34/40 = 85%) switched to the trill song. Only one male (1/34 = 2.9%) copulated with- out production of the trill song. D. The female mounts and copulates with the male approximately 20 s after the beginning of the trill song. All females (34 individuals) mounted the male when the male emitted the trill song (although only one male failed to pass the spermatophore). E. When copulation ended, the female dismounts and leaves the male. Illustrations by Yu Hirayama. Table 2. Effect of the temporal structure of the courtship song on copulation success. Results were obtained using generalized linear model testing. Explanatory variable t-value P-value Chirp duration 0.41 0.68 Interval length 0.39 0.70 Dominant frequency 0.55 0.59 No. chirpst 1.04 0.31 No. ticks# 0.47 0.64 Pulse duration 0.59 0.56 Pulse interval 1.42 G17 No. pulse/chirp 1 22 0.23 Tick duration 0.61 i155 Tick interval 0.12 0:91 + Number of chirps between ticks; + Number of ticks between chirps. 253 song for approximately 25 min while the female stays nearby. Most of the pairs had a courtship song lasting approximately 25 min. However, some pairs (12.5%) did not change to the trill song after 40 min, and copulation did not occur. Identifying the factors that lead to the change in the song, such as micro-movements or female chemical cues, is an important area for future research. Specific environments may cause the evolution of novel song types and mating behaviors. For example, owing to the abundance of acoustically orienting parasitoids, two new male morphs have been observed in the Hawaiian populations of the Pacific field cricket Teleogryllus oceanicus (Le Guillou, 1841) producing a novel purring song (Tinghitella et al. 2018, Fitzgerald et al. 2022). Simi- larly, the evolution of long courtship durations and the trill song in D. csikii may be related to habitat characteristics. Alternatively, such songs may evolve through phylogenetic constraints or Fishe- rian arbitrary processes of mate choice independent of environ- mental characteristics. Comparisons of mating behavior with oth- er closely related species, such as D. nigrofasciatus, may be neces- sary, but such research is hindered by a lack of information on the courtship behavior of these species. Future research should also consider whether the mating behaviors reported here are advanta- geous in sandy beach environments such as the habitat of D. csikii. Availability of data The data used for this study are available from the correspond- ing author on request. B 40 30 25 ~, 30 ro 20 D = 20 15 oO © 10 LL «10 5 rr rs a a a | 0 50 100 150 200 250 0 500 1000 1500 2000 2500 Latency to courtship (s) Courtship duration (s) C D 15 30 25 > 920 10 D =S 15 oT a0 5 had Le i 600 0 62006©64006«6©660—Cs 80 200 400 800 Latency to copulation (s) Copulation duration (s) Fig. 6. Histogram of mating behavior parameters of Dianemobius csikii. A. Latency to courtship, B. courtship duration (i.e., time from start of courtship song to change to trill song), C. latency to copula- tion (i.e., time between trill song and copulation), and D. copulation duration. The white bar indicates no copulation within 40 min (B). JOURNAL OF ORTHOPTERA RESEARCH 2024, 33(2) 254 Ethics approval statement All experimental procedures in this study were conducted in accordance with the guidelines for the use of animals in research in Kagoshima University. 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