Embed Size (px)
Citation preview
© 2003 The Netherlands Entomological Society
Entomologia Experimentalis et Applicata
106
: 73–77, 2003
73
Blackwell Science, Ltd
Mate location in the green capsid bug,
Lygocoris pabulinus
Falko P.
Drijfhout
1
*†,
Astrid T.
Groot
2
,
Teris A.
van Beek
1
&
J. Hans
Visser
3
1
Laboratory of Organic Chemistry, Phytochemical Section, Wageningen University, Dreijenplein 8, 6703 HB Wageningen, the Netherlands;
2
Department of Entomology, North Carolina State University, Raleigh NC 27695, USA;
3
Plant Research International, Wageningen UR, PO Box 16, 6700 AA Wageningen, the Netherlands
Accepted: 10 October 2002
Key words
:
Lygocoris pabulinus
, Miridae, GC-EAD, alkenes (
Z
)-9-pentacosene, (
Z
)-7-pentacosene, mate location behavior, sex pheromone, chemical communication, hydrocarbons, attraction
Abstract
Headspace extracts from female
Lygocoris pabulinus
(L.) (Heteroptera: Miridae) as well as female legextracts attracted male
L. pabulinus
in Y-track olfactometer bioassays. In contrast, only female legextracts were active in the vibration bioassay. Male extracts had no activity at all in either bioassay.When the female leg extract was analysed by coupled gas chromatography-electroantennography(
Z
)-9-pentacosene and (
Z
)-7-pentacosene were EAD-active. (
Z
)-9-Pentacosene and sometimes(
Z
)-7-pentacosene were also observed in female headspace extracts. EAG responses could be obtainedfor (
Z
)-9-pentacosene. A mixture of (
Z
)-9-pentacosene and (
Z
)-7-pentacosene in the ratio 5 : 1 elicitedvibration behaviour in males. The results indicate that these alkenes are important cues for male
L. pabulinus
in mate location behaviour.
Introduction
The mating behaviour of the green capsid bug,
Lygocorispabulinus
, has been studied in detail as part of studies toidentify its sex pheromone (Groot et al., 1998a; Groot, 2000).Yet, the identification of the site of production remainsunknown. Both field and laboratory tests showed thatmales are attracted to females (Blommers et al., 1988; Grootet al., 1996, 1998a) and headspace extracts from femaleswere significantly more attractive to males than maleheadspace extracts in a Y-track olfactometer (Drijfhout et al.,2002). GC-EAG recordings on these female headspace extractsshowed that three compounds elicited an EAG response inmales. These three compounds, however, were also foundin male headspace extracts. In addition, male and femaleheadspace extracts were almost identical (Drijfhout et al.,2002).
Although there is ample evidence that males areattracted to females, no specific calling behaviour hasbeen observed in females (Groot et al., 1998a). However, aspecific behaviour of males was discovered, a vibration ofthe abdomen. Hence, a vibration bioassay was developed
to test the activity of different extracts from males andfemales (Groot et al., 1998a, 1998b). The vibrationbehaviour of males towards extracts from female legs aswell as the chemical composition of these extracts hasbeen described by Drijfhout & Groot (2001), showing adistinct difference between the leg extract from femalesand males.
Leg extracts and headspace collections were studied ina Y-track olfactometer, because of the distinct differencebetween male and female leg extracts and the lack ofdifference in the male and female headspace extracts.Additional GC-EAG experiments and vibration bioassayswere done using the leg extracts from females.
Methods and materials
Insects
Lygocoris pabulinus
was reared under summer conditionson potted potato plants, cultivar Bintje, in wooden cages ina greenhouse at 22
±
2
°
C, 65
±
5% r.h., L18:D6, followingthe procedure of Blommers et al. (1997). Every 2–3 daysthe newly emerged adults were collected from the rearingcages, after which the sexes were isolated in separate rearingcages. In this way, virgin males and females of known ageswere continuously available for the experiments (Grootet al., 1998a).
*Correspondence: e-mail: [email protected]†Present address: Department of Entomology, Cornell University, Comstock Hall, Ithaca, NY 14850, USA.
74
Drijfhout
et al.
Bioassays
Olfactometer tests.
The Y-olfactometer used for conductingthese experiments has been described by Visser & Piron(1998), specific adjustments were made according to Grootet al. (2001). Extracts from female and male legs were testedagainst a filter paper loaded with 40
µ
l of the solvent usedin preparing the extracts, pentane or a 2 : 1 mixture ofpentane : ether.
Vibration
bioassays.
This bioassay was carried out asdescribed by Drijfhout & Groot (2001). The filter papers(5 cm diameter) to be tested were loaded with one femaleequivalent of female leg extract or
c
. 70
µ
g of a mixture of(
Z
)-9-pentacosene and (
Z
)-7-pentacosene in a ratio of5 : 1. In a second series, hexyl butyrate and (
E
)-2-hexenylbutyrate (25
µ
g in a ratio of 20 : 1) were added to thealkene mixture.
Sample collection.
Leg extracts from male and female
L. pabulinus
were prepared according to Drijfhout & Groot(2001). The extracts (the legs remained in the solvent) werestored in a freezer (
−
20
±
2
°
C) until used in bioassays.
Coupled gas chromatography-electroantennography (GC-EAD).
Extracts were analysed by a GC-EAD system. AVarian 6000 GC, with two injector ports and a flameionisation detector (FID), was equipped with a DB23 column(40 m
×
0.20 mm ID
×
0.25
µ
m film thickness) (J & WScientific, Folsom, California). Two microlitres of the extractwas injected splitless (splitless time: 0.8 min), injectortemperature was 250
°
C. The initial oven temperature wasset at 50
°
C. After 1 min the oven was programmed at8
°
C min
−
1
to 238
°
C. Hydrogen was used as the carrier gas.The column effluent was split 1 : 1 (using a Y-connector),one connected to the FID and the other to the EAD. Make-up gas (nitrogen, 15 ml s
−
1
) was added just before the splitpoint (also using a Y-connector). The end of the column tothe EAD passed through the second injector-port on theGC (T = 250
°
C) and thereafter through a heating device(T = 250
°
C) and extruded 1–2 mm out of the heatingdevice into a Liebig condenser (45 cm long). Humidifiedand cooled air (
c
. 900 ml min
−
1
), was directed over the
L. pabulinus
antennal preparation. Additionally, cold water(3–7
°
C) was pumped through the Liebig condenser tofurther cool the humidified air so that the temperature atthe antennal preparation was approximately 15
°
C.The antennal preparation was prepared as follows: four
excised male antennae were placed between two gold wires(probe). This probe was subsequently attached to the inputof an amplifier (Syntech, Hilversum, the Netherlands). Theend of each gold wire was spoon-shaped and a drop ofelectrode gel (Spectra 360, Parker Laboratories Inc., USA)
was placed on the spoon part. The tips of the four antennaewere then inserted into the gel thus establishing electricalcontact and holding them in place. Hereafter, the probewas connected to the amplifier and inserted into the Liebigcondenser so that the antennae were within 5 mm of theend of the column. FID and EAG signals were monitoredsynchronously using software and a GC/EAD interfacecard from Syntech.
Chemicals.
Hexyl butyrate was purchased from Roth(Karlsruhe, Germany). (
E
)-2-hexenyl butyrate was synthesizedaccording to Drijfhout et al. (2000). (
Z
)-9-pentacosene and(
Z
)-7-pentacosene were synthesized according to Drijfhout& Groot (2001).
Chemical analysis.
The analysis of extracts from male andfemale legs has been described previously (Drijfhout &Groot, 2001). The headspace extracts were analysed by gaschromatography and coupled GC/MS (Drijfhout et al., 2002).
Statistical analysis.
Results from the Y-track olfactometerand the vibration bioassay were analysed by the softwareprogram SAS (version 8), procedure GENMOD for logisticregression (SAS Institute Inc., 1997). The following com-parisons were made: A1
−
response towards the controlversus response towards each of the tested extracts in theY-track olfactomter. A2
−
response towards the female legextract versus response towards the female headspace extract(data obtained from Drijfhout et al., 2002). B1
−
responsetowards each of the tested extracts or chemicals in thevibration bioassay.
Results
The response of males towards male and female legextracts in the Y-track olfactometer is shown in Table 1.Significantly more males walked towards the female legextracts than towards the blank filter paper. For the maleleg extract, as many males walked towards the control asto the extract. There was no significant difference in theresponse of the males towards the female headspace extract(data from Drijfhout et al., 2002) and towards the femaleleg extract (Table 1).
GC-EAD recordings of the female leg extracts show that(
Z
)-9-pentacosene and (
Z
)-7-pentacosene were EAD-active.Approximately 50% of the males exhibited the sex-specificvibration behaviour when a mixture of these two alkenes,(
Z
)-9-pentacosene and (
Z
)-7-pentacosene at a ratio of 5 : 1,was introduced in the bioassay (Table 2). Addition of thetwo most obvious volatiles present in the female leg extract,hexyl butyrate and (
E
)-2-hexenyl butyrate at a ratio of 20 : 1(Drijfhout & Groot, 2001), did not affect the percentage of
Mate location in the green capsid bug
75
males vibrating. There was no significant difference betweenthe female leg extract and the mixture of (
Z
)-9-pentacoseneand (
Z
)-7-pentacosene. Extracts from male legs wereinactive.
Figure 1 shows that female headspace extracts containsmall amounts of (
Z
)-9-pentacosene and (
Z
)-7-pentacosene.Additional GC-EAD recordings on these female headspaceextracts in this set-up revealed that besides hexyl butyrate,(
E
)-2-hexyl butyrate, and (
E
)-4-oxo-hexenal (Drijfhoutet al., 2002), (
Z
)-9-pentacosene was also EAD-active.
Discussion
The chemical composition of the leg extracts from malesand females has been previously described (Drijfhout &Groot, 2001). The current study shows that a mixture of(
Z
)-9-pentacosene and (
Z
)-7-pentacosene (5 : 1) elicitsvibration behaviour in male
L. pabulinus
. During earlierresearch (Drijfhout & Groot, 2001) no activity was foundusing these alkenes, although some activity was foundwhen male legs were loaded with a five times higher amountof (
Z
)-9-pentacosene than naturally present in female legs.The amounts used in this study were 10–15 times higherthan in the natural samples, but the release rate of (
Z
)-9-pentacosene and (
Z
)-7-pentacosene from legs and filterpaper is unknown. Little is known about the actual releaserate of these rather non-volatile compounds, and it maytherefore be necessary to use higher amounts in bioassays(Schiestl et al., 2000). The subtle difference between male
and female headspace extracts concerned the presence of asmall amount of (
Z
)-9-pentacosene and (
Z
)-7-pentacosenein female headspace extracts (Drijfhout et al., 2002; Figure 1);and these two compounds differed between male and femaleleg extracts (Drijfhout & Groot, 2001). (
Z
)-9-pentacoseneand (
Z
)-7-pentacosene were both EAD-active in female legextracts, but only (
Z
)-9-pentacosene elicited EAG responsesin GC-EAD recordings of female headspace extracts, whichmay be due to the small amounts of (
Z
)-7-pentacosene inthese extracts. Earlier GC-EAD recordings (Drijfhout et al.,2002) did not show any EAG responses to these alkenes,probably due to the long distance (15 cm) between the endof the column and the antennal preparation used. In thealternative set-up this distance was very small (maximumof 5 mm), reducing condensation of these alkenes beforereaching the antennae.
Alkenes or alkadienes have different functions in insectcommunication (Howard & Blomquist, 1982; Blomquistet al., 1987; Lockey, 1988; Howard, 1993; Singer, 1998;Paulmier et al., 1999). Alkenes are mating stimulantpheromones, e.g., (
Z
)-9-pentacosene in the little housefly (Uebel et al., 1977), contact pheromones or short rangeattractants (Carlson & Beroza, 1973; Uebel et al., 1978;Connor et al., 1980; Schiestl et al., 1999, 2000).
In
L. pabulinus
, these two alkenes probably do not onlyelicit vibration behaviour, but might also act as a shortrange attractant, because the female leg extract attracted asmany males as the female headspace extract in the Y-trackolfactometer (Table 1). The presence of the alkenes can
SourceResponse of malestowards extract
Response of males towards control
Female headspace extract1 130 49 *2 a3
Female leg extract 93 37 * aMale headspace extract1 41 42 ns bMale leg extract 33 33 ns b
1Data from Drijfhout et al. (2002).2Significance in each group. *P < 0.001; ns P > 0.3.3Different letters indicate significant differences between groups (P < 0.001).
Table 1 Responses of male L. pabulinus towards male and female leg and headspace extracts in the Y-track olfactometer
SourceFraction of males responding ± SE n
(1) Female leg extract 0.53 ± 0.28 201 a1
(2) (Z)-9-pentacosene + (Z)-7-pentacosene (5 : 1) 0.52 ± 0.17 83 a(3) (Z)-9-pentacosene + (Z)-7-pentacosene (5 : 1) + hexyl butyrate and (E)-2-hexenyl butyrate (20 : 1) 0.43 ± 0.17 30 a(4) Male leg extract 0.03 ± 0.05 33 b
1Different letters indicate significant differences (P < 0.001).
Table 2 Male vibration response to male and female L. pabulinus leg extracts and synthetic compounds
76 Drijfhout et al.
explain why extracts from female legs and headspace extractsare equally attractive to males, while the absence of thesealkenes, or an off-ratio of these compounds may accountfor lack of attraction to male extracts. Olfactometer testswith a mixture of the two active alkenes are needed toelucidate whether they attract males either alone or incombination with volatile compounds present in headspaceextracts.
The sex pheromone compounds of the mirid Campy-lomma verbasci are similar to those found in the headspacefrom male and female L. pabulinus, although the headspacefrom male and female C. verbasci showed a clear and distinctdifference (Smith et al., 1991; Drijfhout et al., 2000).Furthermore, C. verbasci does not show vibration behaviour.Although L. pabulinus may be closely related to C. verbasciwith respect to their life cycle, they may differ with respectto their sexual communication. The courtship behaviourof other bugs has been studied (e.g., Dunbar, 1972; Borgeset al., 1987; Rodriguez & Eberhard, 1994; Wang & Millar,1997), but little in relation to the identification of their sexpheromones. Despite the numerous chemicals that havebeen identified from true bugs (Aldrich, 1988; McBrien &Millar, 1999), knowledge about the role of these chemicalsother then an attractant is sparse.
According to these and earlier results, the sexualcommunication in L. pabulinus might be divided in twodistinct steps: a long-range (Blommers et al., 1988; Grootet al., 1998a), and a close-range orientation (Groot et al.,1998b; Groot, 2000; Drijfhout 2001; Drijfhout & Groot,2001). The strong EAG responses from males compared tothe female response (Drijfhout et al., 2002) suggest thatmales may use volatiles to locate conspecific L. pabulinus.Once they are in the vicinity of other L. pabulinus theymight use (Z)-9-pentacosene and (Z)-7-pentacosene tolocate the females or to distinguish between males andfemales. Field studies are necessary to investigate the role
of (Z)-9-pentacosene and (Z)-7-pentacosene, and othercompounds on male attraction.
Acknowledgements
We thank Anneke Heijboer for rearing the green capsidbug, Dr M. A. Posthumus for kindly providing gold for theprobe used in the GC-EAD experiments, and Dr R. Gortfor performing the statistical analyses. This research wassupported by the Technology Foundation (NWO/STW) ofthe Netherlands (WBI44.3383).
References
Aldrich JR (1988) Chemical ecology of Heteroptera. AnnualReview of Entomology 33: 211–238.
Blommers L, Bus V, de Jongh E & Lentjes G (1988) Attraction ofmales by virgin females of the green capsid bug, Lygocorispabulinus (Heteroptera: Miridae). Entomologische BerichtenAmsterdam 48: 175–179.
Blommers LHM, Vaal FWNM & Helsen HHM (1997) Lifehistory, seasonal adaptations and monitoring of common greencapsid Lygocoris pabulinus (L.) (Hemiptera: Miridae). Journalof Applied Entomology 121: 389–398.
Blomquist GJ, Nelson DR & de Renobales M (1987) Chemistry,biochemistry and physiology of insect cuticular lipids. Archivesof Insect Biochemistry and Physiology 6: 227–265.
Borges M, Jepson PC & Howse PE (1987) Long-range matelocation and close-range courtship behaviour of the green stinkbug, Nezara viridula and its mediation by sex pheromones.Entomologia Experimentalis et Applicata 44: 205–212.
Carlson DA & Beroza M (1973) Field evaluation of (Z)-9-tricosene, a sex attractant pheromone of the house fly.Environmental Entomology 2: 555–559.
Connor WE, Eisner T, Vander Meer RK, Guerro A, Ghiringelli D& Meinwald J (1980) Sex attractant of an arctiid moth (Uteth-eisa orntrix): a pulse chemical signal. Behavioral Ecology andSociobiology 7: 35–63.
Figure 1 Chromatogram of GC-MS analysis on a DB23 column of headspace extract from female L. pabulinus. Insert: reconstructed ion chromatogram with an m/z-value of 350 amu (M+ of both (Z)-9-pentacosene and (Z)-7-pentacosene).
Mate location in the green capsid bug 77
Drijfhout FP (2001) Studies Towards the Sex Pheromone of theGreen Capsid Bug. PhD Thesis, Wageningen University, TheNetherlands.
Drijfhout FP & Groot AT (2001) Close-range attraction in Lygocorispabulinus (L.). Journal of Chemical Ecology 27: 1133–1149.
Drijfhout FP, Groot AT, Posthumus MA, van Beek TA & de Groot Æ(2002) Coupled Gas Chromatographic–Electroantennographicresponses of Lygocoris pabulinus (L.) to female and maleproduced volatiles. Chemoecology 12: 113–118.
Drijfhout FP, van Beek TA, Visser JH & de Groot Æ (2000)On-line thermal desorption-gas chromatography of intactliving insects for pheromone analysis. Journal of ChemicalEcology 26: 1383–1392.
Dunbar DM (1972) Notes on the mating behavior of Geocorispunctipes (Hemiptera: Lygaeidae). Annals of the EntomologicalSociety of America 65: 764–765.
Groot AT (2000) Sexual Communication in the Green CapsidBug. PhD Thesis, Wageningen University, The Netherlands.
Groot AT, Drijfhout FP, Heijboer A, van Beek TA & Visser JH(2001) Disruption of sexual communication in the mirid bugLygocoris pabulinus by hexyl butanoate. Agricultural andForest Entomology 3: 49–55.
Groot AT, Schuurman A, Judd GJR, Blommers LHM & Visser JH(1996) Sexual behaviour of the green capsid bug Lygocorispabulinus (L.) (Miridae): an introduction. Proceedings ofExperimental and Applied Entomology 7: 249–251.
Groot AT, van der Wal AJ, Schuurman A, Visser JH, Blommers LHM& Van Beek TA (1998a) Copulation behaviour of Lygocorispabulinus under laboratory conditions. Entomologia Experi-mentalis et Applicata 88: 219–228.
Groot AT, Visser JH, Geerts R, Drijfhout FP & van Beek TA(1998b) Behavourial response of Lygocoris pabulinus (Heteroptera:Miridae) to potential sex pheromone compounds. Proceedingsof Experimental and Applied Entomology 9: 191–195.
Howard RW (1993) Cuticular hydrocarbons and chemicalcommunication. Insect Lipids: Chemistry, Biochemistry andBiology. (ed. by D W Stanley-Samuelson & D R Nelson),pp. 179–226. University of Nebraska Press, Lincoln and Lincoln.
Howard RW & Blomquist GJ (1982) Chemical ecology andbiochemistry of cuticular hydrocarbons. Annual Review ofEntomology 27: 149–172.
Lockey KH (1988) Lipids of the insect cuticle: origin, compositionand function. Comparative Biochemistry and Physiology B 98:595–645.
McBrien HL & Millar JG (1999) Phytophagous bugs. Pheromonesof Non-Lepidopterous Insects Associated with AgriculturalPlants. (ed. by J Hardie & A K Minks), pp. 277–304. CABIPublishing, UK.
Paulmier I, Bagnères A, Afonso CMM, Dusticier G, Rivière G &Clément J (1999) Alkenes as a sexual pheromone in the leaf-cutter bee Megachile rotundata. Journal of Chemical Ecology25: 471–490.
Rodriguez RL & Eberhard WG (1994) Male courtship before andduring copulation in two species of Xyonysius bugs (Hemipetra,Lygaeidae). Journal of the Kansas Entomological Society 67:37–45.
SAS Institute Inc. (1997) The GENMOD Procedure SAS/STAT®Software: Changes and Enhancements Through Release 8.0,pp. 249–348. SAS Institute Inc., Cary, NC.
Schiestl FP, Ayasse M, Paulus HF, Löfstedt C, Hansson BS,Ibarra F & Francke W (1999) Orchid pollination by sexualswindle. Nature 399: 421–422.
Schiestl FP, Ayasse M, Paulus HF, Löfstedt C, Hansson BS,Ibarra F & Francke W (2000) Sex pheromone mimicry inthe early spider orchid (Ophrys sphegodes): patterns ofhydrocarbons as the key mechanism for pollination bysexual deception. Journal of Comparative Physiology A 186:567–574.
Singer TL (1998) Roles of hydrocarbons in the recognitionsystems of insects. American Zoologist 38: 394–405.
Smith FS & Pierce HD Jr & Borden JH (1991) Sex pheromone ofthe mullein bug, Campylomma verbasci (Meyer) (Heteroptera:Miridae). Journal of Chemical Ecology 17: 1437–1447.
Uebel EC, Schwars M, Sonnet PE, Miller RW & Menzer RE (1978)Evaluation of the mating stimulant pheromone of Fanniacanicularis, F. pusio and F. femoralis as attractants. FloridaEntomologist 61: 139–143.
Uebel EC, Sonnet PE, Menzer RE, Miller RW & Lusby WR (1977)Mating-stimulant pheromone and cuticular lipid constituentsof the little house fly, Fannia canicularis (L.). Journal ofChemical Ecology 3: 269–278.
Visser JH & Piron PGM (1998) An open y-track olfactometer forrecording of aphid behavioural responses to plant odours.Proceedings of Experimental and Applied Entomology 9:41–45.
Wang Q & Millar JG (1997) Reproductive behavior of Thyantapallidovirens (Heteroptera: Pentatomidae). Annals of theEntomological Society of America 90: 380–388.
