Mating behaviour and evidence for sex-specific pheromones in hedypathes betulinus (coleoptera: cerambycidae: lamiinae)

Mating behaviour and evidence for sex-specific pheromones inHedypathes betulinus (Coleoptera: Cerambycidae: Lamiinae) Laborato´rio de Semioquı´micos, Departamento de Quı´mica, Universidade Federal do Parana´, Curitiba-PR, Brazil cerambycidae, mating behaviour,pheromones, Y-olfactometer The mating behaviour and evidence for sex-specific pheromones of themain pest of green mate, Ilex paraguariensis, of the southern region of Brazil, Hedypathes betulinus (Klug, 1825) (Coleoptera: Cerambycidae: Lamiinae), were studied in the laboratory. The mating sequence of Laborato´rio de Semioquı´micos, Departamento H. betulinus includes: female approaching the male, antennal contact, de Quı´mica, Universidade Federal do Parana´, male mounting the female and start of copulation. This mating sequence CP 19081, CEP 81531-990, Curitiba-PR, Brazil.
E-mail: [email protected] provided support for our hypothesis that recognition of males by femalesmay be mediated by a male-produced pheromone and males recognize Received: October 2, 2008; accepted: April females by contact pheromones. The behavioural responses in an olfac- tometer of male and female H. betulinus to volatiles released by maleand female conspecifics confirmed the presence of a male-produced sex pheromone. This finding suggests that visual cues are not primary ineliciting a sexual response from females. Three male-specific compoundswere detected in the chromatographic analysis, providing chemical sup-port to the behavioural data. Males did not respond to dead-washedfemales but 70% of males attempted to mate with dead-washed extract-treated females, suggesting that extracts contained contact pheromones.
Based on these results, a male-produced sex pheromone and a female-produced contact pheromone should be essential in the communicationsystem of H. betulinus.
act over short and/or long distances for mate loca- tion (Schlyter and Birgersson 1999; Allison et al.
Cerambycid beetles are among the most important insect pests worldwide, degrading lumber and damag- Green mate, Ilex paraguariensis St. Hil., is the eco- ing and killing trees in forests, orchards, plantations nomic base of several municipalities in the southern and urban landscapes. The lack of information on region of Brazil. One of the biggest problems faced behaviour of adults, particularly the role of semio- by its producers is the control of insect pests, being chemicals in reproduction, hinders development of Hedypathes betulinus (Klug, 1825) the main one effective detection and management strategies for many cerambycid species that are economically The adults measure approximately 25 mm of length and have white coloured body with dark In most cerambycid beetles, it has been considered spots; they present long and thin antennae with that mate location depends on males encountering alternated dark and white spots. During oviposition, females by chance, and that recognition of females is the female places only one egg in each branch. The by antennal contact or in response to very short- larvae are white, apod and bore into and feed on range pheromones that only operate over distances the branches and trunks of green mate. They build of several centimetres (Hanks 1999). In some ceram- longitudinal galleries into the branches, which stop bycids, there is evidence of volatile pheromones that the normal circulation of the sap, resulting in mori- Sex-specific pheromones in Hedypathes betulinus bund plants. As the bore progresses, the larvae leave height · 3.5 cm outside diameter) and volatiles emit- sawdust behind it, which accumulates in the base of ted were trapped on 0.8 g of Super Q (Alltech, Deer- the stalk, accusing its presence. The larvae can field, IL) columns as previously reported (Zarbin destroy 60% of green mate plants (Branda˜o Filho et al. 2003), and collected daily for 24 h over 15 1945; Mazuchowski 1991; Cassanello 1993), there- consecutive days. A pushed humidified and charcoal fore, two management strategies, collecting manu- filtered airstream (1 l/min) was maintained through ally the adults and uprooting infested branches and the aeration apparatus. Each aeration chamber was trunks, intend to prevent infestation.
connected to a glass adsorbent trap (11 cm long · Knowledge of H. betulinus mating behaviour may 1 cm diameter) containing Super Q. Volatiles were provide information for the development of moni- eluted from Super Q with distilled hexane, changing toring and control measures. This study was under- the adsorbent traps each 10 collections. The daily taken to investigate mating behaviour sequence and extracts were not combined and were concentrated to search for sex-specific pheromones in H. betulinus.
to 400 ll (one insect per 100 ll) under an argonstream (Zarbin et al. 1999; Zarbin 2001).
Adult insects used in all experiments were collected Response of H. betulinus to volatiles from either sex directly from green mate crops located in Sa˜o Ma- was tested in a binary choice Y-tube olfactometer, teus do Sul, Parana, Brazil in December 2006 using humidified, charcoal filtered air at a rate of through May 2007 and transferred to the laboratory.
4 l/min. Hydrochloric acid and ammonium hydrox- They were sexed according Cassanello (1993), who ide were mixed to visualize the plume distribution described that males have thicker antennae scape inside the system (Baker and Linn 1984). The olfac- and femur fore legs than females. Males and females tometer consisted of a Y-shaped glass tube 4 cm in were held separately in plastic boxes (7.5 cm diame- diameter with a 40-cm long main tube and two 20- cm long arms. Odour sources consisted of a 2 · 2 cm branches, and maintained at 25 Æ 2°C, 60 Æ 5% RH piece of filter paper loaded with 20 ll of the extract and a photoperiod of 12 : 12 L : D. The mating his- (0.2 insect equivalent) of either male or female vola- tory of field-captured beetles was unknown.
tile extract or hexane (control), placed into the baseof either arm of the olfactometer. One male orfemale was introduced into the base of the main tube of the olfactometer and their behaviour was In the field, H. betulinus mating has been observed observed for 20 min. Beetles that walked upwind during photophase, mainly between 2:00 and 6:00 and made direct contact with the filter paper that p.m. (Guedes et al. 2000). To describe the mating contained the odour source (insect extract or con- behaviour sequence of H. betulinus and identify cues trol) within 20 min was recorded as a response. A in mate location, we observed beetles in laboratory beetle that did not walk upwind to any odour source arenas during the same time period. Mating pairs within 20 min was recorded as no response. After (n = 8) were placed with sexes 30 cm apart in individ- every four insects tested, the Y-tube was cleaned ual plastic containers (38 cm length · 27 cm width).
with alcohol and left to dry for 5 min and the posi- For each pair, we recorded how sexes approached each other until the final mating. The mating behav- between odour sources to avoid any positional bias.
iour of beetles was recorded using a digital camera (HP Previous test showed that there was no difference in Photosmart M627; Hewlett-Packard, Miami, FL, USA) choice made either by male or female in the olfac- to facilitate analysis. We studied videotapes of beetles tometer when both of the arms were blank, indicat- to determine whether one sex showed directed move- ing that there was no a positional bias in the ments towards the other from a distance.
olfactometer. Four experiments were conducted: (i)response of males to male extract vs. control;(ii) response of females to male extract vs. con- trol; (iii) response of male to female extract vs.
Groups of four males and females, separated by sex, control; and (iv) response of female to female extract were placed in glass aeration chambers (33 cm vs. control. For each experiment, we tested at least Sex-specific pheromones in Hedypathes betulinus 50 individuals and each individual was tested once Responses of males to dead female and dead- only. Experiments were performed during the fourth washed extract-treated females were compared to to 10th hour of photophase when adult beetles were their response to dead-washed females using a chi- typically active (Guedes et al. 2000).
squared test, program BioEstat 3.0 (Ayres et al.
Data on the response of males and females to dif- ferent treatments (male extract, female extract vs.
control) were analysed by the chi-squared test using BioEstat 3.0 (Ayres et al. 2003). Insects that did notmake a choice were excluded from statistical analysis.
approach of the female to the male, antennal con-tact, mounting and copulation. All couples observed Gas chromatographic (GC) analyses were performed Approach: Females within 30 cm of a males, always on a Varian 3800 GC equipped with flame ionization approached the motionless males, not the reverse detector, electronic pressure control, and operated in (stages 1 and 2, fig. 1), suggesting that female was splitless mode. A VA-5 (30 m · 0.25 m · 0.25 lm) responding to either visual or chemical cues pro- capillary column was used under the following ana- lytical conditions: initial temperature of 50°C for No pre-mounting courtship display by H. betulinus 1 min with an increase 7°C/min until 250°C, which males was observed (stages 1 and 2, fig. 1). Similarly was maintained for 10 min. Upon finishing, thechromatograms obtained with extracts of female andmales were analysed for the presence of sex-specificcandidate pheromone components.
This test was performed following the proceduredescribed by Ginzel and Hanks (2003) and Ginzelet al. (2003). Individual females were freeze killed()4°C for 30 min), allowed to warm to room temper-ature ($15 min), and presented to a male in a plasticarena (15 cm diameter · 18 cm tall) to test whethermales would recognize dead females and attempt tomate, demonstrating that recognition cues wereintact.
females were removed by immersing them in three 1-ml aliquots of hexane for 10 min each; aliquots werecombined and concentrated to 1 ml under an argonstream. Dead-washed females were then presentedindividually to the same male to see if that male nolonger responded, demonstrating that chemical cueshad been removed and mating was not elicited bymechanoreception alone. To prove that the extractcontained the pheromone, we pipetted 1 ml femaleextract on each original dead-washed female, allowedit to evaporate, and presented the female again tomales to see if the recognition cue was restored. Atrial was scored as a ‘response’ if the male, afterantennal contact, mounted her and attempted to Fig. 1 Mating behaviour in Hedypathes betulinus. 1 – Female and copulate. Non-response males showed none of theses male from distance of 30 cm; 2 – female approaches male; 3 – female behaviours or continued to walk after first contacting touches male antennae with her antennae; 4 – male mounts the the female. Each of 10 dead females was presented to female, rotating 180°; 5 – male clasps the female pronotum or elytra with his forelegs; 6 – copulation.
Sex-specific pheromones in Hedypathes betulinus to Oemona hirta (Wang and Davis 2005) and many Table 1 Responses of individual male and female Hedypathes betuli- other cerambycids (Hanks 1999). (ii) Antennal con- nus adults to male/female extracts in Y-tube olfactometer tact: the female walked towards the male and touched his antennae with her antennae (stage 3,fig. 1). (iii) Mounting: immediately after antennal contact, the male mounted the female, rotating180°, and clasped her pronotum or elytra with his forelegs (stages 4 and 5, fig. 1). Males showed no interest in females until antennal contact, suggesting that chemical recognition of females is involved. (iv)Copulation: The male bends his abdomen to connect Statistically significant differences, chi-squared test, P < 0.05.
the female genitalia, and extracts her ovipositor byextending his hind legs (stage 6, fig. 1).
All females were receptive and usually immobile example, male-produced sex pheromones have been during copulation or slowly walked short distances reported in Monochamus alternatus (Fauziah et al.
and did not move their antennae. A similar behav- 1987), Anaglyptus subfasciatus (Nakamuta et al. 1994), iour was observed in Neoclytus mucronatus mucronatus, Hylotrupes bajulus (Fettko¨ther et al. 1995), Anoplopho- Megacyllene caryae and Megacyllene robiniae (Ginzel ra glabripennis (Zhang et al. 2002), Steirastoma breve and Hanks 2003). After mating, males H. betulinus (Liendo et al. 2005), Anoplophora malasiaca (Fukaya guarded females by remaining on their back. This et al. 2005) and Xylotrechus quadripes (Hall et al.
behaviour was observed in the field for the same species by Guedes et al. (2000). Mate guarding is male-produced pheromone in Tetropium fuscum and Tetropium cinnamopterum, the first evidence in the Anoplophora chinensis (Wang et al. 1996b) and Anoplophora glabripennis (Lance et al. 2003; More- The chromatographic profiles of volatiles from wood et al. 2004). Male mate-guarding has usually male and female H. betulinus are shown in fig. 2, and been explained as competition for fertilization of clearly indicate the existence of three male-specific more eggs by fending off rival males (Wang et al.
compounds (a–c), providing chemical support to the These results of the mating sequence provided Furthermore, the mating sequence in H. betulinus support for our hypothesis that recognition of males by females may be mediated by either a male- approached females directly until the latter touched produced pheromone or vision and that males them with their antennae (fig. 1). This reliance on recognize females by contact pheromones.
antennal contact is apparently common among longhorned beetles (Michelsen 1966; Hughes 1981; females were significantly attracted to odours from Akutsu and Kuboki 1983; Kim et al. 1992; Hanks male volatile extract when compared to a hexane et al. 1996; Wang et al. 1996a; Lingafelter 1998; Dejia control: 36 females chose the male extract and eight et al. 1999; Hanks 1999) and is consistent with recog- nition by contact pheromones (Kim et al. 1993; P < 0.01); however, males were not attracted to Fukaya et al. 1996, 2000; Wang 1998; Ginzel et al.
odours from male extract: 18 males chose the male 2003). In the same manner, the use of palpi in extract and 14 males chose the control (v2 = 0.50; mating recognition is probably involved in mate recognition of some cerambycids (Fukaya and Honda (v2 = 0.23; d.f. = 1; P = 0.75) nor males (v2 = 0.39; 1992; Ibeas et al. 2008). For example, males of d.f. = 1; P = 0.68) were attracted to odours from M. alternatus (Kim et al. 1992), A. malasiaca (Fukaya female volatile extract (table 1). The attraction of et al. 2000; Akino et al. 2001), Psacothea hilaris (Fukaya et al. 1996, 1997), Xylotrechus colonus (Ginzel evidence that the mating sequence H. betulinus is et al. 2003), Neoclytus mucronatus mucronatus, Mega- initially mediated by a male-produced sex phero- cyllene caryae, M. robiniae (Ginzel and Hanks 2003), Dectes texanus texanus (Crook et al. 2004), Prionus cali- Sex pheromones are thought to be the most fornicus (Barbour et al. 2007), Monochamus galloprovin- important cue for mate recognition in cerambycid cialis (Ibeas et al. 2008, 2009) display behaviour that beetles (Hanks 1999; Allison et al. 2004). For suggest the use of contact pheromones.
Sex-specific pheromones in Hedypathes betulinus Fig. 2 Gas chromatographic analysis of vola-tiles obtained from male and female Hedypa-thes betulinus adults, showing three male-specific compounds, peaks a, b and c.
In the arena bioassays, all 20 males attempted to Agricultura, CAPES and Fundac¸a˜o Arauca´ria de mate with dead females (v2 = 20; d.f. = 1; P < 0.001) Amparo a` Pesquisa do Estado do Parana´. The authors (N = 10) but did not respond to the same female also wish to thank the Editors and the anonymous after they had been washed in hexane, suggesting referees for the valuable comments and suggestions.
the evidence that females produce a cuticular con-tact pheromone which had been removed by the solvent, and that recognition was not cued solely bymechanoreception.
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