Mate Searching in Ennya Maculicornis (Membracidae: Polyglyptini) Initiated by Females: Behavioural and Acoustic Descriptions

Ecological Entomology (2019), DOI: 10.1111/een.12718

Mate searching in Ennya maculicornis (Membracidae: Polyglyptini) initiated by females: behavioural and acoustic descriptions

ROMINA COSSIO-RODRIGUEZ,1,2 REGINALD B. COCROFT,3 , HERMANN M. NIEMEYER1 and CARLOS F. PINTO1 4 1Departamento de Ciencias Ecológicas, Universidad de Chile, Facultad de Ciencias, Santiago, Chile, 2Facultad de Ciencias y Tecnología, Universidad Mayor de San Simón, Cochabamba, Bolivia, 3Biological Sciences, University of Missouri–Columbia, Columbia, Missouri, U.S.A. and 4Facultad de Tecnología, Universidad Mayor Real y Pontificia de San Francisco Xavier de Chuquisaca, Sucre, Bolivia

Abstract. 1. In treehoppers in which courtship has been studied, males initiate the search for females by periodically emitting a vibrational signal. The responses by the female are used by males as a beacon and give rise to a duet. 2. Courtship and mating of the treehopper Ennya maculicornis were characterised through the simultaneous recording of vibrational signals and the behaviour of males and females in an arena. 3. In E. maculicornis, female initiated mate searching. Females produced two types of signals during the this process: (i) a signal that preceded the approach by the male and (ii) a signal that preceded mating. Males emitted two signals associated with two stereotyped body movements: (i) a signal produced as a response to the first signal emitted by the female, involving a change in the male’s locomotory mode and the approach to the female, and (ii) a signal produced after finding and holding on to the female, involving simultaneous abdomen raising and wing fluttering. These signals were repeated several times before the female emitted the second signal. The four signalling patterns were observed in all recordings in which mating was observed. When any of the signals was missing, mating did not occur. 4. Female-biased sex ratios in E. maculicornis, along with iteroparity, are suggested to explain the initiation of mate searching behaviour by females. A comparison of data with that from other treehoppers indicates that vibrational signals and their associated behaviour are more diverse among treehoppers than has been appreciated previously. Key words. Courtship behaviour, Ennya maculicornis, female mate search, vibrational signals, substrate-borne signals.

Introduction (Liker et al., 2013; Fritzsche et al., 2016) and the occurrence of polyandry or polygyny (Clutton-Brock, 2007). In some The search for potential mating partners is more frequently instances, information may be exchanged during courtship initiated by males than by females (Trivers, 1972; Andersson, that takes the form of duets of signals (e.g. visual, chemical, 1994; Andersson & Simmons, 2006; Clutton-Brock, 2007). The vibrational) emitted by males and females, eventually leading examples in which females initiate mate searching have been to the choice of a mating partner (Rodríguez & Barbosa, 2014). related to the energy that she invests in offspring (Magrath & Insects use different types of signals to attract a partner Komdeur, 2003; Campbella et al., 2009), a bias in sex ratio for mating and during courtship. For example, visual traits may be used to attract the attention of a potential mate Correspondence: Carlos F. Pinto, Laboratorio de Química Ecológ- (Harari & Brockmann, 1999; Li et al., 2017), chemical stim- ica, Departamento de Ciencias Ecológicas, Facultad de Ciencias, uli (pheromones) may play an important role in communication Universidad de Chile, Casilla 653, Santiago, Chile. during courtship and mating (Sass, 1983; Kawazu & Tatsuki, E-mail: [email protected] 2002; Mazor & Dunkelblum, 2005; Sadek et al., 2012; Groot,

2014; Ma et al., 2017), and airborne (i.e. Orthoptera: Gryllidae) inside a cooler to a laboratory in Cochabamba city to perform or substrate-borne (i.e. Hemiptera: Membracidae) acoustic sig- video and audio recordings. Plants of S. maternum were trans- nals may be emitted to locate and attract a potential partner or to planted from Incachaca to a greenhouse and used for rearing signal willingness to mate (Gerhardt & Huber, 2002; Greenfield, of nymphs. Because nymphs molted asynchronously and adults 2002; Cocroft, 2010). become sexually receptive around 12 days after becoming In treehoppers (Membracidae: Hemiptera), males have adults, experiments were performed using adults of similar age been observed to start courtship in all cases investigated to (2 weeks). Adults were sexed and marked with a permanent date: Enchenopa binotata (Rodríguez et al., 2012), Tylopelta marker (Sharpie®; Newell Company, Ogdensburg, New York) gibbera (Legendre et al., 2012), Umbonia crassicornis (De using the same code to mark individuals of the same group to Luca, 2015; Miles et al., 2017), Spissistilus festinus (Hunt, avoid placing individuals from the same plant (potential sib- 1993), Ennya chrysura (Miranda, 2006), Guayaquila sp. lings) in the same recording arena. Individuals of each sex were (R. Cossio-Rodríguez, unpublished data), Vanduzea arquata kept separately on different plants enclosed by a tulle sleeve. (R. B. Cocroft, unpublished data) and Publilia reticulata (R. L. Rodríguez, unpublished data). Attraction of a partner is achieved mainly through vibrational signals. The signals travel along Signal recording the plant substrate and receptive females respond with other Vibrational signals of E. maculicornis were recorded in the vibrational signals, starting a duet (Miranda, 2006; Rodríguez laboratory using a phono cartridge held firmly with a mini tripod & Cocroft, 2006; Legendre et al., 2012; Rodríguez et al., 2012). (Joby GorrillaPod; Vitec Imaging Distribution, U.K.) to avoid Preliminary laboratory recordings in the treehopper Ennya movements that could affect the recording of the signals. The maculicornis (Membracidae: Polyglyptini) showed that females needle of the phono cartridge lightly touched the main stem of . emit the first courtship vibrational signals In the present study, the plant close to its base. The cartridge was connected to a we describe the acoustic and behavioural patterns of courtship direct box amplifier (ULTRA-DI DI100; Behringer, Germany) and mating in this species and also characterise the vibrations and subsequently to an audio recorder (LS-100; Olympus, produced by both sexes and their potential ecological role. Japan). Parallel video recordings were performed using a digital camera (HDR-CR160; Sony Corp., Japan). Vibrational and video signals were subsequently aligned using power director Materials and methods 12 (Cyberlink, Taiwan). Study site Characterisation of courtship behaviour Fieldwork was performed at Incachaca (Cochabamba, Bolivia: ∘ ′′ ∘ ′′ 17 15 Sto6549 W; 2359 m a.s.l.), a cloud forest within In total, 46 recordings were made with E. maculicornis on the Yungas biogeographical province receiving more than plants of S. maternum. Two types of bioassays were performed: 3700 mm of rain per year (Navarro & Maldonado, 2002). The (i) with one female and one male on a plant (n = 25) and (ii) host plant of E. maculicornis at this locality is the endemic with more than one female and/or more than one male on a species Solanum maternum Bohs & Nelson, 1997 (Solanaceae) plant (n = 21): two females with two males (n = 8); two females (Caceres-Sanchez et al., 2017). with one male (n = 6) and three females with two males (n = 7). Males and females were placed on two opposing leaves of the plant as distant as possible from each other (approximately Study species 15 cm). Temperature and relative humidity were measured at the start of the recording with a thermometer–hygrometer The life cycle of E. maculicornis comprises three stages: (i) (model 4465CF; Extech Instruments, Boston, Massachusetts) egg; (ii) five nymphal instars; and (iii) adult. Adults show sexual placed approximately 7 cm away from the plant stem, with the dimorphism in both size (females are usually larger than males) average temperature and relative humidity being 21.1 ± 2.3 ∘C and the colour of their pronotum (black in males and green in and 47.8 ± 8.8%, respectively. Simultaneous characterisation females but turning to yellow as females age) (Caceres-Sanchez of behavioural traits (video) and recordings of vibrational et al., 2017). Females show some degree of maternal care, signals produced by E. maculicornis allowed the description mostly during the egg stage and the first week after hatching of courtship and mating. Recording of bioassays was ended if (Caceres-Sanchez et al., 2017). Females may oviposit more there was no activity after 60 min of recording, if there was no than once, sometimes side by side with females of Ennya response by the male within 90 min of the first signal emitted chrysura Fairmaire (Membracidae), in which case nymphs form by a female, or a few minutes after copulation began because mixed-species aggregations (Caceres-Sanchez et al., 2017). no signals were produced during copulation by either female Individuals of E. maculicornis were collected as fifth-instar or male. nymphs to ensure that the adults used in the experiments did not have courtship experience. In total, 10 field trips were performed. In each trip, two groups of 10 nymphs were collected, Acoustic and statistical analyses each group from a different individual plant with pure E. maculicornis cohorts. Individuals within a group were very likely Vibrational signals were analysed using raven pro, version siblings. Nymphs were transported on a twig of the host plant 1.5 (Cornell Laboratory of Ornithology, Ithaca, New York).

The variables measured from the spectrogram were the duration of signals and the intervals between signals. The dominant frequency, the fundamental frequency, and the upper and lower frequencies, as measured at −10 dB relative to the dominant frequency, were determined from the power spectrum. A sampling rate of 44 100 Hz and an fft size of 4096 were used. Fisher’s exact 2 × 2 tests were used to evaluate whether female signalling was associated with a higher probability of the male responding, whether the courtship signal of males was associated with an increased likelihood of an F-2 signal of females and whether the occurrence of all signals (two from the male, two from the female) was associated with the occurrence of mating. Welch’s t-test was used to compare the durations of female and male signals.

Results

Mate search and courtship behaviour in E. maculicornis

Courtship patterns in both types of bioassays were similar: when more than two individuals were present, the courtship Fig. 1. interaction was established between a single female and a single F-1 signal emitted by the female at the onset of mate searching. male with no participation of other individuals present in the arena. Hence, recordings from both bioassays were considered type (ii)]; in the other 12 cases (six of each type of bioassay), the in the description of the eight sequential events occurring males did not respond to the signal of the female (i.e. they did not during courtship and mating (detailed descriptions of the signals perform the M-1 signal). In the 23 replicates where females did involved follow the general sequence): (i) initially, the female not emit the F-1 signal [18 replicates in bioassays of type (i) and repeatedly produces a signal, the F-1 signal; (ii) after perceiving five in bioassays of type (ii)], males did not emit the M-1 signal. the F-1 signal, the male starts to move mainly towards the female A comparison of the frequency of occurrence of F-1 signals and with a locomotory mode consisting of short bouts of stamping the subsequent ocurrence of M-1 signals (P < 0.001, Fisher’s on the plant, thereby producing the M-1 signal; (iii) a duet starts exact 2 × 2 test) showed that the F-1 signal is a necessary but where the female repeatedly produces the F-1 signal and the not sufficient condition to induce production of the M-1 signal male produces the M-1 signal when approaching the female; by males. (iv) courtship starts when the male positions himself over the female and holds on to her, raises his abdomen and flutters his wings, producing a characteristic vibrational signal (M-2 The M-1 signal of males. Signal M-1 (Fig. 2) had one com- signal); (v) the female responds with aggressive behaviours ponent, with a duration of 0.27 ± 0.14 s, with intervals of (kicking and shaking her body) when the male holds on to 1.18 ± 0.60 s, and a fundamental frequency of 71.7 ± 2.9 Hz, her; (vi) the struggle continues with the male continuously an upper frequency of 153 ± 3.5 Hz and a dominant frequency producing the M-2 signal; (vii) the female emits a final complex of 110.3 ± 2.9 Hz. A duet was then established with F-1 signals signal, the F-2 signal; and (viii) the male stops abdomen raising from the female and M-1 signals from the male (Fig. 3). and wing fluttering (and hence stops producing M-2 signals), the female stops producing the F-2 signal, and copulation takes place. Copulation lasts from one to several hours. The M-2 signal and courtship movements by males. M-2 signals with a single component appear as long vertical lines in the spectrogram and oscillogram and a single compo- Characterisation of vibrational signals emitted during nent (Fig. 4), with a duration of 0.19 ± 0.026 s, with intervals courtship of 4.72 ± 2.35 s, and a lower frequency of 67 ± 3.2 Hz, an upper frequency of 130 ± 48 Hz and a dominant frequency of The F-1 signal of females. Signal F-1 (Fig. 1) is harmonically 97.2 ± 2.9 Hz. In 10 of the 11 cases where the male courted the structured with a single component (measures are presented female, she accepted the male and mating occurred, suggesting as the mean ± SD): duration of 0.74 ± 0.05 s, with intervals that courtship by the male is important for acceptance by the between signals of 0.70 ± 0.08 s, a fundamental frequency of female (occurrence of courtship by the male versus occurrence 70.8 ± 2.1 Hz, an upper frequency of 128.6 ± 3.7 Hz and a of the F-2 signal, P < 0.001, Fisher’s exact 2 × 2 test). dominant frequency of 97.9 ± 2.4 Hz. This signal was emitted in 23 of the 46 replicated bioassays. After F-1 was emitted, males performed the M-1 signal in 11 cases and mated in all of these The F-2 signal of females. In all bioassays where mating cases [one replicate in bioassays of type (i) and 10 in bioassays of occurred, the F-2 signal was emitted repeatedly by the female

Fig. 2. M-1 signal emitted by the male as a response to the F-1 signal Fig. 4. The M-2 signal emitted by the male when it raises its abdomen emitted by the female. and flutters its wings producing vibrations that are registered as vertical lines.

male signals. Additionally, female signals were longer than male signals in all cases (F-1 versus M-1: t = 7.328, d.f. = 650, P < 0.001; F-1 versus M-2: t = 3.028, d.f. = 389, P = 0.002; F-2 versus M-1: t = 69.003, d.f. = 94, P < 0.001; F-2 versus M-2: t = 61.464, d.f. = 121, P < 0.001).

Mating success. When every one of the behaviours and vibrational signals described was performed, mating occurred (Fig. 6); if any of the behaviours or signals was absent, the couple did not mate (mating success versus the occurrence of the complete mating sequence, P < 0.0001, Fisher’s exact 2 × 2 test). Two replicates showed atypical results: in one case, the male courted the female for approximately 90 min and the female never emitted the F-2 signal. During that time, the male never tried to mate and, after that time, the male stopped performing courtship movements and freed the female. In another case, after being accepted, the male tried to mate with the female but was not successful in coupling his genitalia with hers. Fig. 3. Duet consisting of F-1 signals by the female and M-1 signals by the male. Discussion before the mating event (P < 0.001, Fisher’s exact 2 × 2 test). Females of E. maculicornis initiated mate searching by emitting This signal has two components (Fig. 5) and had a mean duration the F-1 signal and showed their final acceptance of the male of 1.2 ± 0.3 s, with intervals of 2.8 ± 0.12 s, a lower frequency by producing the F-2 signal. Because the F-1 signal is emitted of 52.9 ± 4.9 Hz, an upper frequency of 108.6 ± 5.7 Hz and a spontaneously and is a necessary condition to involve the dominant frequency of 77.4 ± 5.7 Hz. male in the mate searching sequence, it may correspond to a ‘calling’ signal. On the other hand, because the F-2 signal was produced in all bioassays where mating occurred and no mating Comparison between female and male signals. On average, occurred if the signal was not produced, it may correspond to an female signals were more complex (had more components) than ‘acceptance’ signal.

a vibrational signal; in all cases, the female may respond and perform a duet with the calling male and, eventually, the male further emits a second signal before mating (Hunt, 1993; Miranda, 2006; Legendre et al., 2012; Rodríguez et al., 2012; De Luca, 2015; Miles et al., 2017). Thus, females of these membracids emit a single type of vibrational signal before copulating. By contrast, females of E. maculicornis emit two types of signals: the calling and acceptance signals. This is the first report female membracids emitting two types of vibrational signals with different meanings to the male. The use of two different signals by the female during mate searching is not without precedent in other animal groups; for example, females of the leafhopper Homoladisca vitripennis emit one vibrational signal in response to calling males and another before mating occurs (Nieri et al., 2017). Vibrational signals produced by females have mostly been reported to be simpler (fewer number of components) and shorter than male signals, particularly within the order Hemiptera (Rodríguez & Barbosa, 2014.). By contrast, in the case of E. maculicornis, vibrationals signals of females were longer and more complex than those of males. This is yet another Fig. 5. The F-2 signal emitted by the female subsequent to the feature in which E. maculicornis differs from other membracids. courtship signal M-2 by males. Although vibrations produced by prey may be used by arthropod predators and parasitoids to locate them (Brownell & Farley, The male responded to the female’s F-1 signal with the M-1 1979a,b,c; Pfannenstiel et al., 1995; Casas et al., 1998; Barth, signal and, after he held the female, with the M-2 signal, thus 2002; Devetak, 2014), females of some species appear to have expressing his choice. On the other hand, females expressed evolved to produce short signals to avoid detection by predators, their choice by emitting the F-2 acceptance signal after the male especially in sites with high predator abundance (Greenfield, emits the M-2 signal. In 23 bioassays where females emitted 2002; Bailey, 2003; Rodríguez & Cocroft, 2006). This situation the call signal, males responded in only 11 of them, whereas, in would have been expected in E. maculicornis because its host only one case where males emitted the M-2 signal, the female plant has a high richness and abundance of predators (unpub- did not respond with the F-2 signal and mating did not occur; lished data). However, E. maculicornis females emit two long this pattern suggests that some males may not have reached and complex signals compared with those produced by males. sexual maturity at the age used or also the possibility that This may correspond to a trade-off in which the females could males can choose or reject a female based on their call. These be exposed to predators, at the same time being more efficient observations suggest that both females and males express their in finding a mate. Interestingly, the acceptance signal produced choice through vibrational signals in insects. (Claridge, 1985; by the female, once she has accepted a mating partner, is longer Cocroft & McNett, 2006; Hill, 2009). and is repeated more frequently than the initial calling signal. In the membracid species that have been studied, it is the Cases in which the initial attraction of a potential partner relies male who starts mate searching, in particular, by first emitting on female signalling are more common in scenarios where males

Fig. 6. Flowchart with the mate searching process. Numbers above arrows denote the frequency with which an event occurred.

© 2019 The Royal Entomological Society, Ecological Entomology, doi: 10.1111/een.12718 6 Romina Cossio-Rodriguez et al. invest more energy in their offspring (Magrath & Komdeur, RCR wrote the paper, with continuous revision by CFP, HMN 2003; Campbella et al., 2009); however, this is not the case in and RBC. E. maculicornis because males do not provide parental care. A study of the biology and ecology of the same population of E. References maculicornis indicated the presence of a higher proportion of females (Caceres-Sanchez et al., 2017), which is an attribute that Andersson, M. (1994) Sexual Selection. Princeton University Press, can also promote the development of female strategies to initiate Princeton, New Jersey. attraction towards males through competition to find a mate Andersson, M. & Simmons, L.W. (2006) Sexual selection and mate choice. Trends in Ecology & Evolution, 21, 296–302. (Kvarnemo & Simmons, 1999; Clutton-Brock, 2009). A reversal Bailey, W.J. 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Science, of historical, genetic and ecological factors. 318, 1882–1885. Clutton-Brock, T. (2009) Sexual selection in females. Animal Behaviour, 77, 3–11. Acknowledgements Cocroft, R.B. (2010) Vibrational Communication. Encyclopedia of Animal Behavior, pp. 498–505. Academic Press, Oxford, U.K. We thank ISP (International Science Program at Uppsala Uni- Cocroft, R.B. & McNett, G.D. (2006) Vibratory communication in treehoppers (Hemiptera: Membracidae). Insect Sound and Communica- versity) through its BOL-01 and LANBIO (Latin American Net- tion (ed. by S. Drosopoulos and M. Claridge), pp. 305–317. CRC work for Research on Bioactive Natural Compounds) programs, Press, Boca Raton, Florida. as well as IFS (International Foundation for Science) D/5472-1 De Luca, P.A. (2015) Mass correlates with increased mating success for grant, for financial support. We are also indebted to INTE- older but not younger males in thornbug treehoppers. 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(2002) Acoustic Communication in Insects CFP, RCR, RBC and HMN proposed and designed the and Anurans: Common Problems and Diverse Solutions. University study. RCR and CFP performed fieldwork and data gathering. of Chicago Press, Chicago, Illinois.

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