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Two different vibratory signals in Rhodniusprolixus (Hemiptera: Reduviidae)
Acta Tropica 77 (2000) 271 – 278 www.elsevier.com/locate/actatropica
Two different vibratory signals in Rhodnius prolixus (Hemiptera: Reduviidae) G. Manrique *, P.E. Schilman 1 Departamento de Cs. Biolo´gicas, Facultad de Ciencias Exactas y Naturales, Uni6ersidad de Buenos Aires, Ciudad Uni6ersitaria, 1428 Buenos Aires, Argentina Received 16 July 2000; received in revised form 27 July 2000; accepted 14 August 2000
Abstract In this study the substrate-borne stridulatory vibrations produced by Rhodnius prolixus females were recorded and analysed in two different behavioural contexts. In the context of sexual communication females spontaneously stridulated to reject copulatory attempts performed by males. These male-deterring stridulations were fully effective: out of 61 attempts, no copulation occurred. These stridulations consisted of short series of repetitive syllables, each one composed by a single chirp. In the context of defensive behaviour, bugs stridulated if they were clasped or restrained. These disturbance stridulations consisted of long series of repetitive syllables, each one composed by a series of short chirps and a long one. Male-deterring and disturbance stridulations differed in their temporal pattern and frequency spectra, having a main carrier frequency of about 1500 and 2200 Hz, respectively. As no differences in the inter-ridge distances along the whole stridulatory organ were found, the differences in the frequency between both signals could be explained on the basis of a different velocity of rubbing of the proboscis against the prosternal stridulatory organ. It was found that R. prolixus and the related species Triatoma infestans rubbed only the central region of the stridulatory groove (around 1/3 of the total length) to produce disturbance stridulations. The results are discussed in relation to previous work on vibrational sensitivity in R. prolixus and are also compared with results reported for T. infestans. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Rhodnius prolixus; Stridulation; Sexual behavior; Vibratory communication; Defensive behavior; Mechanoreception
1. Introduction Rhodnius prolixus and Triatoma infestans (Hemiptera, Reduviidae, Triatominae) are haema* Corresponding author. Fax: +54-11-45763384. E-mail address: [email protected] (G. Manrique). 1 Present address: Theodor-Boveri-Institut, Lehrstuhl fu¨r Verthaltensphysiologie und Soziobiologie der Universita¨t, Am Hubland, D 97074 Wu¨rzburg, Germany.
tophagous bugs of medical importance as vectors of the flagellate Trypanosoma cruzi responsible for Chagas Disease in South America (Zeledo´n and Rabinovich, 1981). Vibrational communication has been observed in many arthropod species (Markl, 1983). Triatomine bugs are not an exception: they stridulate by rubbing the tip of the proboscis against the striated cuticular prosternal groove (Schofield, 1977; Di Luciano, 1981). Such
0001-706X/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 1 - 7 0 6 X ( 0 0 ) 0 0 1 4 6 - 7
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G. Manrique, P.E. Schilman / Acta Tropica 77 (2000) 271–278
stridulations usually take place when bugs are prevented from moving freely as a response to mechanical disturbance, e.g. while clasping them with forceps (Moore, 1961; Schofield, 1977). In addition, Manrique and Lazzari (1994) found that non-receptive T. infestans females spontaneously produce stridulatory vibrations to reject the copulatory attempts performed by males. Both kind of signals, i.e. disturbance and male-deterring stridulations, were measured in T. infestans as substrate-borne signals and comparatively analysed (Roces and Manrique, 1996). Differences in their temporal pattern, as well as in their frequency spectra, were found, thus suggesting that they have different meanings related to their different behavioural contexts. Unfortunately, no studies on sensitivity to substrate-borne signals in T. infestans have been made. Autrum and Schneider (1948), working with R. prolixus, quantified the sensitivity of vibrational receptors to different frequencies. They measured a minimum threshold at 400 Hz and found no electrical responses of leg nerves to higher frequencies. In the present study the stridulatory signals of R. prolixus were analysed in the context of mating and defensive behaviour. Results are discussed in relation to the vibrational sensitivity reported by Autrum and Schneider (1948), and compared to those reported for T. infestans. In addition, the inter-ridge distances of the stridulatory groove of R. prolixus were measured in order to discuss their relationship with the emission frequency of the stridulations, as well as to compare results between both species. The relationship between the number of pulses recorded and the number of ridges rubbed by both species were also analysed.
1996). Considering that substrate vibrations (rather than airborne) constitute the true signal in terrestrial bugs (Gogala, 1984), an accelerometer was used. In this way, vibrations produced by stridulating R. prolixus females propagated through the insect’s body and substrate reaching the accelerometer. The accelerometer generated a signal which voltage was proportional to the instantaneous acceleration of the moving object. The electrical signals were then amplified, monitored by an oscilloscope and stored in the sound track of a videotape. The behaviour of the bugs was simultaneously videotaped. To record stridulations in the sexual context, in each assay a male and a female were placed into a receptacle where they could freely interact. Twenty-six couples were used. During the interactions each female stridulated between 1 and 18 times, reaching a total of 61 stridulations (12 couples). Thirty-two stridulatory sequences performed by three females were reliably recorded in this way and selected for the analysis. Stridulations as a response to mechanical disturbance were obtained by handling each female with forceps and recorded by pressing its thorax on the accelerometer (N= 31 stridulatory sequences performed by two females, 17 and 14 sequences each one). Vibrational signals were further analysed using an A/D converter (CED 1401, Science Products) and analysing software (CED Spike 2, Science Products). Experiments were done at 25°C and at a light intensity of 25 lx.
2. Materials and methods Adult R. prolixus males and females (2 months after adult emergence) were used throughout the study. They were reared in the laboratory at 28°C and fed every 15 days ad libitum on heparinized bovine blood using an artificial feeder (Nu´n˜ez and Lazzari, 1990). To record the stridulatory signals the device shown in Fig. 1 was used (Roces and Manrique,
Fig. 1. Experimental set-up used to record substrate-borne stridulatory vibrations produced by R. prolixus.
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begins with a jump or mounting of the male on the female, followed by contact of the genitalia of both sexes. Male-deterring stridulations were fully effective. In every case, females rejected the 61 copulatory attempts by stridulating as soon as they were physically contacted by the male (N= 12 couples) and no copulations occurred. During female stridulation, the male was observed either to remain immobile on the female for a few seconds, or to give up after mounting and leave. The males did not stridulate during the assays.
Fig. 2. Prosternal stridulatory organ of a R. prolixus female. Note the transverse ridges along the whole organ.
As previously established for T. infestans, the differences in the main carrier frequency for maledeterring and disturbance stridulations could be explained by either: (1) a difference in the velocity of rubbing of the same region of the groove; or (2) by rubbing the groove at the same velocity but in different regions (Roces and Manrique, 1996). In order to rule out the latter alternative, the inter-ridge distances of the prosternal stridulatory organ of R. prolixus were measured (Fig. 2). Stridulatory organs (N = 3) were cleared overnight in a 10% solution of NaOH at 60°C and thereafter the preparations were mounted in Canada balsam. In order to depict the variability in the ridge density, three areas were arbitrarily defined along the groove. The inter-ridge distances of the stridulatory grooves were measured under a light microscope. In addition, magnified (400 ×) close-up videofilms of disturbance stridulations were recorded to observe which region of the prosternal groove was rubbed.
3. Results As in many reduviids (Lima et al., 1986; Rojas et al., 1990; Rojas and Cruz-Lopez, 1992; Manrique and Lazzari, 1994), mating of R. prolixus
Fig. 3. Male-deterring stridulations produced by a female over time, recorded as an acceleration of the substrate (see experimental set-up). Calibration bars (time in s and acceleration in cm/s2) are indicated. Open arrows indicate copulation attempts of the male prior to its retreat. Signals were amplified 2000 times and filtered between 300 and 4000 Hz.
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Fig. 4. Disturbance stridulations produced by restrained females over time, recorded directly as an acceleration of its dorsal cuticle (see Section 2). Calibration bar (time in s and acceleration in cm/s2) are indicated. Signals were amplified 500 times and filtered between 300 and 4000 Hz.
Fig. 3 shows the male-deterring stridulations and the associated occurrence of male jumping, copulatory attempts and the moment in which the male leaves the female. Male-deterring stridulations consist of short series of repetitive syllables, each one composed of only one chirp. A repetition of four consecutive syllables (four chirps), as well as a magnification of a single syllable is presented in Fig. 3 (below). The chirps were separated by an inter-syllable interval of 23.69 5.3 ms (mean9S.E., N =13, k = 3); this means that male-deterring stridulations were repeated at a rate of 35.69 8.2 syllables per second (mean9 S.E., N = 32 stridulatory sequences, k = 3).
Disturbance stridulations are presented in Fig. 4. They consist of long series of repetitive syllables. In this case, each syllable is composed of a series of short chirps (5.459 0.68, mean9 S.E., range 1–19) and a long one. The mean syllable duration of disturbance stridulations was far longer than that of male-deterring ones: the former reached a value of 124.398.3 ms (mean9 S.E., N=31, k= 2) whilst the latter reached 33.49 7.3 ms (mean9 S.E., N= 22, k= 3). The short and long chirps of disturbance stridulations differed in duration, short-chirps lasted 3.89 0.3 ms (mean9S.E.), considerably less than the long chirp, which lasted 39.6 9 1.6 ms (mean9S.E.). On the other hand, both had similar amplitude: 3.79 1.3 cm/s2 (mean9 S.E.) for the short chirps and 4.19 0.4 cm/s2 (mean9 S.E.) for the long one. The pause between short chirps was 11.59 1.2 ms (mean9 S.E.) and the pause between the last short chirp and the long one was 13.2 91.6 ms (mean 9 S.E.). Considerable differences were found between male-deterring and disturbance stridulations in the inter-syllable interval or repetition rate; the first parameter reached a value of 23.59 5 ms (mean9 S.E.) for male-deterring and 32.294.8 ms (mean9 S.E.) for disturbance stridulations, averaging a repetition rate of 35.69 8.2 syllables per second (mean9S.E., N= 32 sequences, k= 3 females) for male-deterring and 6.590.5 (mean9 S.E., N= 29 sequences, k= 2 females) for disturbance stridulations. The frequency spectra of both kinds of stridulations are presented in Fig. 5. The maximum vibration energy was found in the frequency range of 1400–1600 Hz for male-deterring stridulations, with an harmonic of minor energy at 3500 Hz (Fig. 5, lower plot). The maximum vibration energy for disturbance stridulations was concentrated in the range of 1700–3000 Hz (Fig. 5, top and middle plots). The main carrier frequency was found around 1500 Hz for maledeterring, and 2200 Hz (long chirp) and 2700 Hz (taking into account the whole interval of short chirps) for disturbance stridulations.
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The total number of ridges counted in the stridulatory groove of R. prolixus was around 90. No significant differences in the inter-ridge distances were found between the three areas arbitrarily defined along the groove (Table 1). In the close-up videofilms it was found that R. prolixus and T. infestans rubbed only the central region of their stridulatory groove (around 1/3 of the total length) to produce disturbance stridulations.
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Table 1 Mean (9S.D.) distances between the ridges of the prosternal stridulatory organ of R. prolixus females (see Fig. 2)a Part of the groove
Inter-ridge distance (mm)
Range (mm)
N (k)
Anterior third Middle third Posterior third
7.4 9 0.8 8.1 9 0.5 8.2 9 0.5
5.7–8.6 6.9–9.2 6.9–9.2
76 (3) 77 (3) 74 (3)
a In order to depict the variability in the ridge density, three regions were arbitrarily defined along the groove and between 22 and 27 distances were measured. Data obtained on three females.
4. Discussion
Fig. 5. Frequency spectra of both male-deterring (lower plot) and disturbance stridulations (top and middle plots). Twelve recordings were averaged for male-deterring stridulations and 44 for disturbance stridulations (20 for long and 24 for short chirps) recorded on the thorax. Time windows of 100 ms were used for the Fast Fourier Transform analysis. Signals were filtered between 300 and 4000 Hz. FFT size: 256. Bin width: 195.3 Hz.
In the present work, mating behaviour of R. prolixus followed the general model described for other reduviids (Lima et al., 1986; Rojas et al., 1990; Rojas and Cruz-Lopez, 1992; Manrique and Lazzari, 1994). Briefly, the male jumped onto or mounted the female and then they juxtaposed their genitalia. Although one did not state the conditions of mating success, as indicated for T. infestans (Manrique and Lazzari, 1994), female R. prolixus responded with a rejection behaviour as soon as they were physically contacted by the males that attempted to copulate. This is the first time that vibratory signals in sexual and disturbance contexts have been quantified for R. prolixus as substrate-borne signals. In addition, it has been demonstrated that R. prolixus females spontaneously stridulate to reject the copulatory attempts performed by males. The results are similar to those obtained for T. infestans (Manrique and Lazzari, 1994; Roces and Manrique, 1996). However, some differences between the two species deserve further comment. First, there was a shift to higher frequency values of male-deterring stridulations in R. prolixus (1500 Hz), compared to those of T. infestans (800 Hz). Another evident interspecific difference is that in R. prolixus, both kinds of stridulations consisted of series of repetitive syllables shorter than those of T. infestans. Regarding disturbance stridulations, in T. infestans each syllable was composed by two chirps while in R. prolixus each syllable was composed by a series of short chirps
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and a long one, but the duration of these stridulations were similar in both species, being a little longer in the latter. Concerning male-deterring stridulations, each syllable was composed of one and two chirps in R. prolixus and T. infestans, respectively, but their duration was approximately the same. Furthermore, it is important to mention that in spite of these interspecific differences between the signals, in both species male deterring stridulations have a higher repetition rate and lower frequencies than disturbance stridulations. Moreover, results showed that the inter-ridge distances of the prosternal stridulatory organ of R. prolixus had no differences along the whole organ (Table 1). For this reason, the differences found in the carrier frequency of both signals could be explained by the fact that the bugs rub the same region of the stridulatory organ but at a different velocity. The groove of the related species T. infestans possesses approximately 160 ridges and the number of pulses found in the long chirp during disturbance stridulations were around 80 – 100 (Roces and Manrique, 1996). In that study it was assumed that there was a 1:1 ratio between the number of pulses recorded and the number of ridges rubbed during disturbance stridulations. In R. prolixus, it was found that during long chirps, the number of pulses recorded were approximately 90 (e.g. Fig. 4). Considering the 1:1 ratio occurrence for R. prolixus, these insects should use the whole groove to produce 90 pulses. Magnified close up videofilms of disturbance stridulations of T. infestans and R. prolixus showed that both species rubbed only the central region of the groove, which involved around 1/3 of the total length. Therefore, the 1:1 ratio between the number of pulses recorded and the number of rubbed ridges did not occur. This demonstrates that at least during disturbance stridulations more than one pulse is generated when a ridge is rubbed with the tip of the proboscis, both in T. infestans and R. prolixus. Regarding the possible receptors involved, Hemiptera bugs do not have true subgenual organ (Debaisieux, 1935, 1938), but do have distal scoloparia of the tibia and campaniform sensilla. These organs appear to participate in the reception of
substrate-borne vibrations. It is known that high frequency signals (e.g. \ 1 kHz) suffer an enormous attenuation during propagation through air. One solution to avoid this physical problem would be the use of a higher density substrate to propagate these signals (Michelsen, 1983). That is the case of many insects that communicate each other by vibratory signals through the branches of their host plants (Roces et al., 1993; Coˆkl et al., 1999), or through direct contact vibrations when females reject copulatory attempts performed by males (e.g. Markl et al., 1977; Roces and Manrique, 1996). In particular, it has been demonstrated that some land bugs use the vibratory channel for communication (Gogala and Coˆkl, 1983; Gogala, 1984, 1985). In addition, there are no tympanal organs described for triatomines, and so far there is no behavioural evidence that these bugs respond to air-borne sounds (Schofield, 1977). Thus, substrate-borne vibrations are good candidates to play a role in intraspecific communication in triatomines, as in many other insect species (Markl, 1983). Autrum and Schneider (1948) made electrophysiological recordings to measure the sensitivity of many insects to substrate-borne vibrations of different frequencies. They quantified the electrical responses of the distal scoloparia of the tibia of R. prolixus. These authors did not obtain electrical responses to values higher than 400 Hz for this species. The results imply a sensitivity to frequencies higher than 400 Hz, since R. prolixus females produced vibratory signals of higher frequencies (1400–3000 Hz), and in the case of male-deterring stridulations (1400–1600 Hz), that indeed modified the behaviour of males. One explanation to this contradiction could be that Autrum and Schneider (1948) restricted their measurements only to the distal scoloparia of the tibia, but in fact, receptors localised in other parts of the males’ body could be responsible for the localisation and primary response to the vibratory signal. Candidates for such a role may be located on the antennae, e.g. tricobothria or Johnstons’ organ (Schofield, 1977). This hypothesis is supported by the fact that in the recordings the male’s body was in physical contact with the stridulating female. Therefore, vibrations could
G. Manrique, P.E. Schilman / Acta Tropica 77 (2000) 271–278
reach the antennae directly or propagating through legs and body. Another possible explanation is the presence of different receptor cells for different frequencies, as occurs in the Pentatomidae bug Nezara 6iridula which has different types of neurones that show maximal sensitivity to low or high frequencies (Coˆkl, 1983). The results of disturbance stridulations obtained in the present work agree with the characterisation of insect disturbance stridulations done by Masters (1979, 1980), who considered these signals as defensive responses of insects to predator attacks. In agreement with this, preliminary results obtained for disturbance stridulations from five different species of triatomines evince that most of the vibratory signals have similar frequency values (Manrique and Schilman, 1997). As was previously suggested for stridulations performed by T. infestans (Roces and Manrique, 1996), signals of R. prolixus play a role in the intraspecific communication during sexual context, while disturbance stridulations would be shaped to deter predators. This fact would imply that these latter vibratory signals are not specific, but are a general feature in Triatominae. Morphological studies aimed at identifying, the receptors involved in the reception of vibratory signals, together with electrophysiological studies may allow one to analyse the features of mechanoreception in Triatominae in future investigations.
Acknowledgements The authors are indebted to F. Roces, M. Giurfa, C. Reisenman, A. J. Wainselboim, G.B. Flores, S.A.Minoli, and C.R. Lazzari for critically reading the early version of the manuscript, to C.R. Lazzari, J.A. Nu´n˜ez, and all Insect Physiology Laboratory staff for fruitful discussions, and to three anonymous referees who improved the manuscript with their comments. Research was conducted at the University of Buenos Aires, Argentina, with funding from grants of CONICET to J.A. Nu´n˜ez and from Universidad de Buenos Aires and UNDP/World Bank/WHO (TDR) to C.R. Lazzari. Data analysis was per-
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formed thanks to F. Roces from the University of Wu¨rzburg, Germany. P.E. Schilman and G. Manrique were supported by a Doctoral and Postdoctoral grants from CONICET/Argentina, respectively. The authors are also deeply indebted to DAAD. References Autrum, H., Schneider, W., 1948. Vergleichende Untersuchungen bei den Erschu¨tterungssinn der Insekten. Zeitsch. Vergl. Physiol. 31, 77 – 88. Coˆkl, A., 1983. Functional properties of vibroreceptors in the legs of Nezara 6iridula (L.) (Heteroptera, Pentatomidae). J. Comp. Physiol. 150, 261 – 269. Coˆkl, A., Virant-Doberlet, M., McDowell, A., 1999. Vibrational directionality in the southern green stink bug, Nezara 6iridula (L.), is mediated by female song. Anim. Behav. 58, 1277 – 1283. Debaisieux, P., 1935. Organes Scolopidiaux des Pattes D’Insectes I. Lepidopeteres et Trichopteres. La Cellule 44, 271 – 314. Debaisieux, P., 1938. Organes Scolopidiaux des Pattes D’Insectes II. La Cellule 47, 77 – 202. Di Luciano, V.S., 1981. Morphology of the stridulatory groove of Triatoma infestans (Hemiptera: Reduviidae). J. Med. Entomol. 18, 24 – 32. Gogala, M., 1984. Vibration producing structures and songs of terrestrial Heteroptera as systematic character. Biol. Vestn. 32, 19 – 36. Gogala, M., 1985. Vibrational songs of land bugs and their production. In: Ka¨lmring, K., Elsner, N. (Eds.), Acoustic and Vibrational Communication in Insects. Paul Parey, Berlin, pp. 143 – 150. Gogala, M., Coˆkl, A., 1983. The acoustic behaviour of the bug Phymata crassipes (F.) (Heteroptera). Rev. Can. Biol. Exptl. 42, 249 – 256. Lima, M.M., Ju¨rberg, P., Ribeiro De Almeida, J., 1986. Behaviour of Triatomines (Hemiptera: Reduviidae) vectors of Chagas’ disease. I Courtship and copulation of Panstrongylus megistus (Burm., 1835) in the laboratory. Mem. Inst. Oswaldo Cruz 81, 1 – 5. Manrique, G., Lazzari, C.R., 1994. Sexual behaviour and stridulation during mating in Triatoma infestans (Hemiptera: Reduviidae). Mem. Inst. Oswaldo Cruz 89, 629 – 633. Manrique, G., Schilman, P.E., 1997. Disturbance stridulations in five species of Triatominae. Mem. Inst. Oswaldo Cruz 92 (Suppl. I), 274. Markl, H., 1983. Vibrational communication. In: Huber, F., Markl, H. (Eds.), Neuroethology and Behavioral Physiology. Springer-Verlag, Berlin, pp. 332 – 353. Markl, H., Ho¨lldobler, B., Ho¨lldobler, T., 1977. Mating behavior and sound production in harvester ants (Pogonomyrmex, Formicidae). Ins. Soc. 24, 191 – 212.
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G. Manrique, P.E. Schilman / Acta Tropica 77 (2000) 271–278 blood-sucking bug Triatoma infestans (Hemiptera: Reduviidae). J. Insect Physiol. 42, 231 – 238. Roces, F., Tautz, J., Ho¨lldobler, B., 1993. Stridulation in leaf-cutting ants: short-range recruitment through plantborne vibration. Naturwissenschaften 80, 521 – 524. Rojas, J.C., Cruz-Lopez, L., 1992. Sexual behaviour in two species of Triatoma phyllosoma complex (Hemiptera: Reduviidae) under laboratory conditions. J. Med. Entomol. 29, 13 – 18. Rojas, J.C., Malo, E.A., Gutierrez-Martinez, A., Ondarza, R.A., 1990. Mating behaviour of Triatoma mazzottii Usinger (Hemiptera: Reduviidae) under laboratory conditions. Ann. Entomol. Soc. Am. 83, 598 – 602. Schofield, C.J., 1977. Sound production in some triatomine bugs. Physiol. Entomol. 2, 43 – 52. Zeledo´n, R., Rabinovich, J.E., 1981. Chagas’ disease: an ecological appraisal with special emphasis on its insect vectors. Ann. Rev. Entomol. 26, 101 – 133.
Masters, W.M., 1979. Insect disturbance stridulation: its defensive role. Behav. Ecol. Soc. 5, 187–200. Masters, W.M., 1980. Insect disturbance stridulation: characterization of airborne and vibrational components of the sound. J. Comp. Physiol. 135, 259–268. Michelsen, A., 1983. Biophysical basis of sound communication. In: Lewis, B. (Ed.), Bioacustics, A Comparative Approach. Academic Press, London, pp. 3–38. Moore, T.E., 1961. Audiospectrographic analysis of sounds of Hemiptera and Homoptera. Ann. Entomol. Soc. Am. 54, 273 – 291. Nu´n˜ez, J.A., Lazzari, C.R., 1990. Rearing of Triatoma infestans Klug (Het., Reduviidae) in the absence of a live host. I. Some factors affecting the artificial feeding. J. Appl. Entomol. 109, 87 – 92. Roces, F., Manrique, G., 1996. Different stridulatory vibrations during sexual behaviour and disturbance in the
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Two different vibratory signals in Rhodnius prolixus (Hemiptera: Reduviidae) G. Manrique *, P.E. Schilman 1 Departamento de Cs. Biolo´gicas, Facultad de Ciencias Exactas y Naturales, Uni6ersidad de Buenos Aires, Ciudad Uni6ersitaria, 1428 Buenos Aires, Argentina Received 16 July 2000; received in revised form 27 July 2000; accepted 14 August 2000
Abstract In this study the substrate-borne stridulatory vibrations produced by Rhodnius prolixus females were recorded and analysed in two different behavioural contexts. In the context of sexual communication females spontaneously stridulated to reject copulatory attempts performed by males. These male-deterring stridulations were fully effective: out of 61 attempts, no copulation occurred. These stridulations consisted of short series of repetitive syllables, each one composed by a single chirp. In the context of defensive behaviour, bugs stridulated if they were clasped or restrained. These disturbance stridulations consisted of long series of repetitive syllables, each one composed by a series of short chirps and a long one. Male-deterring and disturbance stridulations differed in their temporal pattern and frequency spectra, having a main carrier frequency of about 1500 and 2200 Hz, respectively. As no differences in the inter-ridge distances along the whole stridulatory organ were found, the differences in the frequency between both signals could be explained on the basis of a different velocity of rubbing of the proboscis against the prosternal stridulatory organ. It was found that R. prolixus and the related species Triatoma infestans rubbed only the central region of the stridulatory groove (around 1/3 of the total length) to produce disturbance stridulations. The results are discussed in relation to previous work on vibrational sensitivity in R. prolixus and are also compared with results reported for T. infestans. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Rhodnius prolixus; Stridulation; Sexual behavior; Vibratory communication; Defensive behavior; Mechanoreception
1. Introduction Rhodnius prolixus and Triatoma infestans (Hemiptera, Reduviidae, Triatominae) are haema* Corresponding author. Fax: +54-11-45763384. E-mail address: [email protected] (G. Manrique). 1 Present address: Theodor-Boveri-Institut, Lehrstuhl fu¨r Verthaltensphysiologie und Soziobiologie der Universita¨t, Am Hubland, D 97074 Wu¨rzburg, Germany.
tophagous bugs of medical importance as vectors of the flagellate Trypanosoma cruzi responsible for Chagas Disease in South America (Zeledo´n and Rabinovich, 1981). Vibrational communication has been observed in many arthropod species (Markl, 1983). Triatomine bugs are not an exception: they stridulate by rubbing the tip of the proboscis against the striated cuticular prosternal groove (Schofield, 1977; Di Luciano, 1981). Such
0001-706X/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 1 - 7 0 6 X ( 0 0 ) 0 0 1 4 6 - 7
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G. Manrique, P.E. Schilman / Acta Tropica 77 (2000) 271–278
stridulations usually take place when bugs are prevented from moving freely as a response to mechanical disturbance, e.g. while clasping them with forceps (Moore, 1961; Schofield, 1977). In addition, Manrique and Lazzari (1994) found that non-receptive T. infestans females spontaneously produce stridulatory vibrations to reject the copulatory attempts performed by males. Both kind of signals, i.e. disturbance and male-deterring stridulations, were measured in T. infestans as substrate-borne signals and comparatively analysed (Roces and Manrique, 1996). Differences in their temporal pattern, as well as in their frequency spectra, were found, thus suggesting that they have different meanings related to their different behavioural contexts. Unfortunately, no studies on sensitivity to substrate-borne signals in T. infestans have been made. Autrum and Schneider (1948), working with R. prolixus, quantified the sensitivity of vibrational receptors to different frequencies. They measured a minimum threshold at 400 Hz and found no electrical responses of leg nerves to higher frequencies. In the present study the stridulatory signals of R. prolixus were analysed in the context of mating and defensive behaviour. Results are discussed in relation to the vibrational sensitivity reported by Autrum and Schneider (1948), and compared to those reported for T. infestans. In addition, the inter-ridge distances of the stridulatory groove of R. prolixus were measured in order to discuss their relationship with the emission frequency of the stridulations, as well as to compare results between both species. The relationship between the number of pulses recorded and the number of ridges rubbed by both species were also analysed.
1996). Considering that substrate vibrations (rather than airborne) constitute the true signal in terrestrial bugs (Gogala, 1984), an accelerometer was used. In this way, vibrations produced by stridulating R. prolixus females propagated through the insect’s body and substrate reaching the accelerometer. The accelerometer generated a signal which voltage was proportional to the instantaneous acceleration of the moving object. The electrical signals were then amplified, monitored by an oscilloscope and stored in the sound track of a videotape. The behaviour of the bugs was simultaneously videotaped. To record stridulations in the sexual context, in each assay a male and a female were placed into a receptacle where they could freely interact. Twenty-six couples were used. During the interactions each female stridulated between 1 and 18 times, reaching a total of 61 stridulations (12 couples). Thirty-two stridulatory sequences performed by three females were reliably recorded in this way and selected for the analysis. Stridulations as a response to mechanical disturbance were obtained by handling each female with forceps and recorded by pressing its thorax on the accelerometer (N= 31 stridulatory sequences performed by two females, 17 and 14 sequences each one). Vibrational signals were further analysed using an A/D converter (CED 1401, Science Products) and analysing software (CED Spike 2, Science Products). Experiments were done at 25°C and at a light intensity of 25 lx.
2. Materials and methods Adult R. prolixus males and females (2 months after adult emergence) were used throughout the study. They were reared in the laboratory at 28°C and fed every 15 days ad libitum on heparinized bovine blood using an artificial feeder (Nu´n˜ez and Lazzari, 1990). To record the stridulatory signals the device shown in Fig. 1 was used (Roces and Manrique,
Fig. 1. Experimental set-up used to record substrate-borne stridulatory vibrations produced by R. prolixus.
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begins with a jump or mounting of the male on the female, followed by contact of the genitalia of both sexes. Male-deterring stridulations were fully effective. In every case, females rejected the 61 copulatory attempts by stridulating as soon as they were physically contacted by the male (N= 12 couples) and no copulations occurred. During female stridulation, the male was observed either to remain immobile on the female for a few seconds, or to give up after mounting and leave. The males did not stridulate during the assays.
Fig. 2. Prosternal stridulatory organ of a R. prolixus female. Note the transverse ridges along the whole organ.
As previously established for T. infestans, the differences in the main carrier frequency for maledeterring and disturbance stridulations could be explained by either: (1) a difference in the velocity of rubbing of the same region of the groove; or (2) by rubbing the groove at the same velocity but in different regions (Roces and Manrique, 1996). In order to rule out the latter alternative, the inter-ridge distances of the prosternal stridulatory organ of R. prolixus were measured (Fig. 2). Stridulatory organs (N = 3) were cleared overnight in a 10% solution of NaOH at 60°C and thereafter the preparations were mounted in Canada balsam. In order to depict the variability in the ridge density, three areas were arbitrarily defined along the groove. The inter-ridge distances of the stridulatory grooves were measured under a light microscope. In addition, magnified (400 ×) close-up videofilms of disturbance stridulations were recorded to observe which region of the prosternal groove was rubbed.
3. Results As in many reduviids (Lima et al., 1986; Rojas et al., 1990; Rojas and Cruz-Lopez, 1992; Manrique and Lazzari, 1994), mating of R. prolixus
Fig. 3. Male-deterring stridulations produced by a female over time, recorded as an acceleration of the substrate (see experimental set-up). Calibration bars (time in s and acceleration in cm/s2) are indicated. Open arrows indicate copulation attempts of the male prior to its retreat. Signals were amplified 2000 times and filtered between 300 and 4000 Hz.
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Fig. 4. Disturbance stridulations produced by restrained females over time, recorded directly as an acceleration of its dorsal cuticle (see Section 2). Calibration bar (time in s and acceleration in cm/s2) are indicated. Signals were amplified 500 times and filtered between 300 and 4000 Hz.
Fig. 3 shows the male-deterring stridulations and the associated occurrence of male jumping, copulatory attempts and the moment in which the male leaves the female. Male-deterring stridulations consist of short series of repetitive syllables, each one composed of only one chirp. A repetition of four consecutive syllables (four chirps), as well as a magnification of a single syllable is presented in Fig. 3 (below). The chirps were separated by an inter-syllable interval of 23.69 5.3 ms (mean9S.E., N =13, k = 3); this means that male-deterring stridulations were repeated at a rate of 35.69 8.2 syllables per second (mean9 S.E., N = 32 stridulatory sequences, k = 3).
Disturbance stridulations are presented in Fig. 4. They consist of long series of repetitive syllables. In this case, each syllable is composed of a series of short chirps (5.459 0.68, mean9 S.E., range 1–19) and a long one. The mean syllable duration of disturbance stridulations was far longer than that of male-deterring ones: the former reached a value of 124.398.3 ms (mean9 S.E., N=31, k= 2) whilst the latter reached 33.49 7.3 ms (mean9 S.E., N= 22, k= 3). The short and long chirps of disturbance stridulations differed in duration, short-chirps lasted 3.89 0.3 ms (mean9S.E.), considerably less than the long chirp, which lasted 39.6 9 1.6 ms (mean9S.E.). On the other hand, both had similar amplitude: 3.79 1.3 cm/s2 (mean9 S.E.) for the short chirps and 4.19 0.4 cm/s2 (mean9 S.E.) for the long one. The pause between short chirps was 11.59 1.2 ms (mean9 S.E.) and the pause between the last short chirp and the long one was 13.2 91.6 ms (mean 9 S.E.). Considerable differences were found between male-deterring and disturbance stridulations in the inter-syllable interval or repetition rate; the first parameter reached a value of 23.59 5 ms (mean9 S.E.) for male-deterring and 32.294.8 ms (mean9 S.E.) for disturbance stridulations, averaging a repetition rate of 35.69 8.2 syllables per second (mean9S.E., N= 32 sequences, k= 3 females) for male-deterring and 6.590.5 (mean9 S.E., N= 29 sequences, k= 2 females) for disturbance stridulations. The frequency spectra of both kinds of stridulations are presented in Fig. 5. The maximum vibration energy was found in the frequency range of 1400–1600 Hz for male-deterring stridulations, with an harmonic of minor energy at 3500 Hz (Fig. 5, lower plot). The maximum vibration energy for disturbance stridulations was concentrated in the range of 1700–3000 Hz (Fig. 5, top and middle plots). The main carrier frequency was found around 1500 Hz for maledeterring, and 2200 Hz (long chirp) and 2700 Hz (taking into account the whole interval of short chirps) for disturbance stridulations.
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The total number of ridges counted in the stridulatory groove of R. prolixus was around 90. No significant differences in the inter-ridge distances were found between the three areas arbitrarily defined along the groove (Table 1). In the close-up videofilms it was found that R. prolixus and T. infestans rubbed only the central region of their stridulatory groove (around 1/3 of the total length) to produce disturbance stridulations.
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Table 1 Mean (9S.D.) distances between the ridges of the prosternal stridulatory organ of R. prolixus females (see Fig. 2)a Part of the groove
Inter-ridge distance (mm)
Range (mm)
N (k)
Anterior third Middle third Posterior third
7.4 9 0.8 8.1 9 0.5 8.2 9 0.5
5.7–8.6 6.9–9.2 6.9–9.2
76 (3) 77 (3) 74 (3)
a In order to depict the variability in the ridge density, three regions were arbitrarily defined along the groove and between 22 and 27 distances were measured. Data obtained on three females.
4. Discussion
Fig. 5. Frequency spectra of both male-deterring (lower plot) and disturbance stridulations (top and middle plots). Twelve recordings were averaged for male-deterring stridulations and 44 for disturbance stridulations (20 for long and 24 for short chirps) recorded on the thorax. Time windows of 100 ms were used for the Fast Fourier Transform analysis. Signals were filtered between 300 and 4000 Hz. FFT size: 256. Bin width: 195.3 Hz.
In the present work, mating behaviour of R. prolixus followed the general model described for other reduviids (Lima et al., 1986; Rojas et al., 1990; Rojas and Cruz-Lopez, 1992; Manrique and Lazzari, 1994). Briefly, the male jumped onto or mounted the female and then they juxtaposed their genitalia. Although one did not state the conditions of mating success, as indicated for T. infestans (Manrique and Lazzari, 1994), female R. prolixus responded with a rejection behaviour as soon as they were physically contacted by the males that attempted to copulate. This is the first time that vibratory signals in sexual and disturbance contexts have been quantified for R. prolixus as substrate-borne signals. In addition, it has been demonstrated that R. prolixus females spontaneously stridulate to reject the copulatory attempts performed by males. The results are similar to those obtained for T. infestans (Manrique and Lazzari, 1994; Roces and Manrique, 1996). However, some differences between the two species deserve further comment. First, there was a shift to higher frequency values of male-deterring stridulations in R. prolixus (1500 Hz), compared to those of T. infestans (800 Hz). Another evident interspecific difference is that in R. prolixus, both kinds of stridulations consisted of series of repetitive syllables shorter than those of T. infestans. Regarding disturbance stridulations, in T. infestans each syllable was composed by two chirps while in R. prolixus each syllable was composed by a series of short chirps
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and a long one, but the duration of these stridulations were similar in both species, being a little longer in the latter. Concerning male-deterring stridulations, each syllable was composed of one and two chirps in R. prolixus and T. infestans, respectively, but their duration was approximately the same. Furthermore, it is important to mention that in spite of these interspecific differences between the signals, in both species male deterring stridulations have a higher repetition rate and lower frequencies than disturbance stridulations. Moreover, results showed that the inter-ridge distances of the prosternal stridulatory organ of R. prolixus had no differences along the whole organ (Table 1). For this reason, the differences found in the carrier frequency of both signals could be explained by the fact that the bugs rub the same region of the stridulatory organ but at a different velocity. The groove of the related species T. infestans possesses approximately 160 ridges and the number of pulses found in the long chirp during disturbance stridulations were around 80 – 100 (Roces and Manrique, 1996). In that study it was assumed that there was a 1:1 ratio between the number of pulses recorded and the number of ridges rubbed during disturbance stridulations. In R. prolixus, it was found that during long chirps, the number of pulses recorded were approximately 90 (e.g. Fig. 4). Considering the 1:1 ratio occurrence for R. prolixus, these insects should use the whole groove to produce 90 pulses. Magnified close up videofilms of disturbance stridulations of T. infestans and R. prolixus showed that both species rubbed only the central region of the groove, which involved around 1/3 of the total length. Therefore, the 1:1 ratio between the number of pulses recorded and the number of rubbed ridges did not occur. This demonstrates that at least during disturbance stridulations more than one pulse is generated when a ridge is rubbed with the tip of the proboscis, both in T. infestans and R. prolixus. Regarding the possible receptors involved, Hemiptera bugs do not have true subgenual organ (Debaisieux, 1935, 1938), but do have distal scoloparia of the tibia and campaniform sensilla. These organs appear to participate in the reception of
substrate-borne vibrations. It is known that high frequency signals (e.g. \ 1 kHz) suffer an enormous attenuation during propagation through air. One solution to avoid this physical problem would be the use of a higher density substrate to propagate these signals (Michelsen, 1983). That is the case of many insects that communicate each other by vibratory signals through the branches of their host plants (Roces et al., 1993; Coˆkl et al., 1999), or through direct contact vibrations when females reject copulatory attempts performed by males (e.g. Markl et al., 1977; Roces and Manrique, 1996). In particular, it has been demonstrated that some land bugs use the vibratory channel for communication (Gogala and Coˆkl, 1983; Gogala, 1984, 1985). In addition, there are no tympanal organs described for triatomines, and so far there is no behavioural evidence that these bugs respond to air-borne sounds (Schofield, 1977). Thus, substrate-borne vibrations are good candidates to play a role in intraspecific communication in triatomines, as in many other insect species (Markl, 1983). Autrum and Schneider (1948) made electrophysiological recordings to measure the sensitivity of many insects to substrate-borne vibrations of different frequencies. They quantified the electrical responses of the distal scoloparia of the tibia of R. prolixus. These authors did not obtain electrical responses to values higher than 400 Hz for this species. The results imply a sensitivity to frequencies higher than 400 Hz, since R. prolixus females produced vibratory signals of higher frequencies (1400–3000 Hz), and in the case of male-deterring stridulations (1400–1600 Hz), that indeed modified the behaviour of males. One explanation to this contradiction could be that Autrum and Schneider (1948) restricted their measurements only to the distal scoloparia of the tibia, but in fact, receptors localised in other parts of the males’ body could be responsible for the localisation and primary response to the vibratory signal. Candidates for such a role may be located on the antennae, e.g. tricobothria or Johnstons’ organ (Schofield, 1977). This hypothesis is supported by the fact that in the recordings the male’s body was in physical contact with the stridulating female. Therefore, vibrations could
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reach the antennae directly or propagating through legs and body. Another possible explanation is the presence of different receptor cells for different frequencies, as occurs in the Pentatomidae bug Nezara 6iridula which has different types of neurones that show maximal sensitivity to low or high frequencies (Coˆkl, 1983). The results of disturbance stridulations obtained in the present work agree with the characterisation of insect disturbance stridulations done by Masters (1979, 1980), who considered these signals as defensive responses of insects to predator attacks. In agreement with this, preliminary results obtained for disturbance stridulations from five different species of triatomines evince that most of the vibratory signals have similar frequency values (Manrique and Schilman, 1997). As was previously suggested for stridulations performed by T. infestans (Roces and Manrique, 1996), signals of R. prolixus play a role in the intraspecific communication during sexual context, while disturbance stridulations would be shaped to deter predators. This fact would imply that these latter vibratory signals are not specific, but are a general feature in Triatominae. Morphological studies aimed at identifying, the receptors involved in the reception of vibratory signals, together with electrophysiological studies may allow one to analyse the features of mechanoreception in Triatominae in future investigations.
Acknowledgements The authors are indebted to F. Roces, M. Giurfa, C. Reisenman, A. J. Wainselboim, G.B. Flores, S.A.Minoli, and C.R. Lazzari for critically reading the early version of the manuscript, to C.R. Lazzari, J.A. Nu´n˜ez, and all Insect Physiology Laboratory staff for fruitful discussions, and to three anonymous referees who improved the manuscript with their comments. Research was conducted at the University of Buenos Aires, Argentina, with funding from grants of CONICET to J.A. Nu´n˜ez and from Universidad de Buenos Aires and UNDP/World Bank/WHO (TDR) to C.R. Lazzari. Data analysis was per-
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formed thanks to F. Roces from the University of Wu¨rzburg, Germany. P.E. Schilman and G. Manrique were supported by a Doctoral and Postdoctoral grants from CONICET/Argentina, respectively. The authors are also deeply indebted to DAAD. References Autrum, H., Schneider, W., 1948. Vergleichende Untersuchungen bei den Erschu¨tterungssinn der Insekten. Zeitsch. Vergl. Physiol. 31, 77 – 88. Coˆkl, A., 1983. Functional properties of vibroreceptors in the legs of Nezara 6iridula (L.) (Heteroptera, Pentatomidae). J. Comp. Physiol. 150, 261 – 269. Coˆkl, A., Virant-Doberlet, M., McDowell, A., 1999. Vibrational directionality in the southern green stink bug, Nezara 6iridula (L.), is mediated by female song. Anim. Behav. 58, 1277 – 1283. Debaisieux, P., 1935. Organes Scolopidiaux des Pattes D’Insectes I. Lepidopeteres et Trichopteres. La Cellule 44, 271 – 314. Debaisieux, P., 1938. Organes Scolopidiaux des Pattes D’Insectes II. La Cellule 47, 77 – 202. Di Luciano, V.S., 1981. Morphology of the stridulatory groove of Triatoma infestans (Hemiptera: Reduviidae). J. Med. Entomol. 18, 24 – 32. Gogala, M., 1984. Vibration producing structures and songs of terrestrial Heteroptera as systematic character. Biol. Vestn. 32, 19 – 36. Gogala, M., 1985. Vibrational songs of land bugs and their production. In: Ka¨lmring, K., Elsner, N. (Eds.), Acoustic and Vibrational Communication in Insects. Paul Parey, Berlin, pp. 143 – 150. Gogala, M., Coˆkl, A., 1983. The acoustic behaviour of the bug Phymata crassipes (F.) (Heteroptera). Rev. Can. Biol. Exptl. 42, 249 – 256. Lima, M.M., Ju¨rberg, P., Ribeiro De Almeida, J., 1986. Behaviour of Triatomines (Hemiptera: Reduviidae) vectors of Chagas’ disease. I Courtship and copulation of Panstrongylus megistus (Burm., 1835) in the laboratory. Mem. Inst. Oswaldo Cruz 81, 1 – 5. Manrique, G., Lazzari, C.R., 1994. Sexual behaviour and stridulation during mating in Triatoma infestans (Hemiptera: Reduviidae). Mem. Inst. Oswaldo Cruz 89, 629 – 633. Manrique, G., Schilman, P.E., 1997. Disturbance stridulations in five species of Triatominae. Mem. Inst. Oswaldo Cruz 92 (Suppl. I), 274. Markl, H., 1983. Vibrational communication. In: Huber, F., Markl, H. (Eds.), Neuroethology and Behavioral Physiology. Springer-Verlag, Berlin, pp. 332 – 353. Markl, H., Ho¨lldobler, B., Ho¨lldobler, T., 1977. Mating behavior and sound production in harvester ants (Pogonomyrmex, Formicidae). Ins. Soc. 24, 191 – 212.
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