A Floral-Derived Compound Attractive to the Tephritid Fruit Fly Parasitoid

A Floral-Derived Compound Attractive to the Tephritid Fruit Fly Parasitoid Diachasmimorpha longicaudata (Hymenoptera:Braconidae)Eric Rohrig&John Sivinski&Peter Teal&Charles Stuhl&Martin AlujaReceived:30October2007/Revised:20December2007/Accepted:23January2008#Springer Science+Business Media,LLC2008Abstract Many adult hymenopteran parasitoids,even host-feeding species,consume the nectar of flowering plants. Previous field studies had identified plants attractive (Lobularia maritima L.)and unattractive(Spermacoce verticillata L)to certain opiine braconids(Hymenoptera). Under laboratory conditions,Diachasmimorpha longicau-data(Ashmead),a parasitoid of tephritid fruit fly larvae and representative opiine,responded in flight tunnels to L. maritima but not to S.verticillata.V olatile chemicals of the two flowers were collected and analyzed by using capillary gas liquid chromatography and mass spectral analysis. Acetophenone was isolated from L.maritima but not from S.verticillata.In flight tunnels, D.longicaudata were exposed to10concentrations(doses)of acetophenone. Female parasitoids showed a significant attraction to several acetophenone doses,with concentrations of25and 50ng the most attractive.No odor source,either floral or floral-derived,was attractive to male parasitoids.Reliable trapping systems for parasitoid species,particularly species such as D.longicaudata used for augmentative biological control,would be a valuable monitoring tool.At present, there are few,if any,florally derived synthetic lures for attracting hymenopteran parasitoids.Keywords Acetophenone.Opiinae.Traps.Biological control.FlowersIntroductionUnderstanding the population dynamics of target pests and their natural enemies is critically important in Integrated Pest Management(IPM)(Kogan1998;Suckling et al. 2002).Attraction and trapping techniques are among the simplest means of determining pest and natural enemy distributions and densities in the field,and this information, combined with known insect biology,can help determine what controls should be applied,when and where to use them,and the status of non-target organisms that might be affected(Prokopy1985).Among the controls best timed and placed through the use of trapping-demographic data is the inundative release of beneficial insects(Jewitt and Carpenter2001).It is particularly important to monitor the dispersal and survival of the mass-released predators/para-sitoids so that adjustments can be made to the releases,and their efficacies estimated.At present,most pest-insect attractants are based on sex pheromones,and majority of these are female-derived chemicals that capture males(Foster and Harris1997). Sex pheromones are also emitted by female,and more rarely male,parasitic Hymenoptera(Sivinski and Petersson 1997;Kainoh1999),and can also be used as attractants (Rice and Jones1982;Jewett and Carpenter2001;Suckling et al.2002).However,compounds associated with hosts,orJ Chem EcolDOI10.1007/s10886-008-9438-yE.RohrigDepartment of Entomology and Nematology, University of Florida,Bldg.970,Natural Area Dr., Gainesville,FL32611,USAE.Rohrig:J.Sivinski(*):P.Teal:C.Stuhl USDA-ARS Center for Medical, Agricultural and Veterinary Entomology, 1660/1700SW23rd Dr.,Gainesville,FL32604,USAe-mail:jsivinski@ M.AlujaInstituto de Ecología,A.C.,Apartado Postal63,91000Xalapa,Veracruz,Méxicoenvironments where hosts might be found,may be more generally attractive to a presumably largely non-virgin and sexually unresponsive female parasitoid population.For example,among tephritid fruit fly parasitoids,Fopius arisanus(Sonan)and Diachasmimorpha longicaudata (Ashmead)females are attracted to fruit volatiles(Messing and Jang1992;Eben et al.2000;Altuzar et al.2004),the latter particularly to decaying fruit and associated fungi (Greany et al.1977).Yet another tephritid parasitoid Psyttalia fletcheri Silvestri responds to decaying fruits and leaves of pumpkins and cucumbers(Messing et al.1996). Nishida(1956)earlier found that stem tissues of cucurbits are attractive to P.fletcheri,and Messing et al.(1996) suggest that the attractiveness to leaf and stem tissues may be due in part to“green leaf volatiles”,a suite of common leaf-derived compounds known to be attractive to other braconid species(Whitman and Eller1990).Flowers of many species of plants produce odors that are also highly attractive to various natural enemies(Andersen 1987;Maini and Burgio1999;Ventura et al.2000;Landolt et al.2001).Although nectar feeding,and to a lesser extent pollen consumption,is common in the parasitic Hymenop-tera(Jervis et al.1993),there has been little if any use of floral-derived compounds as attractants.However,there may be advantages to the use of such compounds.Like host odors,but unlike male-produced sex pheromones,flower volatiles might be more consistently appealing to the mated females that likely constitute the bulk of many parasitoid populations.In the course of a previous field comparison,flowers that were attractive(Lobularia maritima L.)and unattrac-tive(Spermacoce verticillata L.)to an economically significant subfamily of Braconidae,the Opiinae,were preliminarily identified(Rohrig2006).In the present experiments,the flight tunnel responses of D.long-icaudata,a widely used tephritid biological control agent and representative opiine,to the two flowering plants and to a compound uniquely identified from L.maritima volatiles were determined.These plants and the model insect are described below.Spermacoce verticillata,“shrubby false buttonweed”(Rubiaceae)is native to the West Indies,but can be found in Florida and Texas as well as west Africa,the tropical Americas,and the south Pacific.Habitats consist of open or disturbed sandy zones and pinelands where it grows as a shrub.Spermacoce verticillata is a perennial dicot whose small white flowers form dense clusters at the upper stem nodes.Flowers have a mean corolla depth of1.5mm,a width of1.0mm,and possess a honey guard at the interior base of the corolla(Sivinski et al.2006).The nectar is a major food source for the mole cricket ectoparasitoids Larra bicolor F.and Larra analis F.(Hymenoptera: Sphecidae)in Florida(Frank and Parkman1999).Lobularia maritima,“alyssum”(Brassicaceae),was introduced from the Mediterranean region and now ranges throughout most of the United States including Hawaii. This plant is a hardy,non-weedy,annual dicot herb that flowers consistently from fall through spring in subtropical areas.Small cruciform-stalked white flowers grow in clusters randomly throughout the plant that attract large numbers of parasitic Hymenoptera(Chaney1998).On average,corollas are0.67mm wide by 1.4mm deep (Sivinski et al.2006).Lobularia maritima nectar increases the longevity of several ichneumonoid parasitoids,both in the laboratory and in the field(Johanowicz and Mitchell2000;Berndt and Wratten2005).While L.maritima flowers increase the longevity and realized fecundity of the egg parasitoid Trichogramma carverae Oatman and Pinto(Begum2004), this is only true of the white flower variety(Begum et al. 2004).This was the variety used in the present experiments.Due to availability and its wide use as a biological control agent,D.longicaudata was chosen to examine the response of a representative opiine to the floral volatile. While a variety of chalcidoids,diapriids,figitids,and ichneumonoids parasitize Tephritidae(e.g.,Sivinski et al. 2000),braconids of the subfamily Opiinae are typically the most numerous and diverse members of the guilds that attack frugivorous species(Purcell1998).Opiinae are solitary,koinobiont,larval/egg-prepupal endoparasitoids of Cyclorrhapha Diptera(Bess et al.1961;Lopez et al.1999; Wharton1999).Several species are considered important regulators of fruit fly populations(Wharton1989)and have been introduced,and frequently established,throughout the world(Ovruski et al.2000).Diachasmimorpha longicaudata is one of the most widely used of these opiine tephritid biological control agents(Ovruski et al.2000).Adult females use their relatively long ovipositor to parasitize a number of second-and third-instar fruit fly species’larvae in a wide variety of host fruits(Wharton1989;Sivinski et al.2000).The species was originally discovered in the Indo-Philippine region where it attacked Bactrocera spp.(Wharton and Marsh1978),and in1947was introduced into Hawaii for the control of oriental fruit fly,Bactrocera dorsalis (Hendel)(Clancy and Dressner1952).In1972,D.long-icaudata was established in Florida to control the Caribbe-an fruit fly,Anastrepha suspensa(Loew),and it subsequently reduced populations by~40%(Baranowski et al.1993).In addition to wide-spread introductions,D.longicau-data has been mass-reared and inundatively released,either alone or in combination with sterile male flies,for the control of Ceratitis capitata(Wong et al.1991),and various Anastrepha spp.(Sivinski et al.1996;Montoya et al.2000).In Florida,such releases suppressed A.suspensaJ Chem Ecolpopulations by as much as96%and have been considered as a means of supporting“fly-free”zones to facilitate citrus exports.Methods and MaterialsDiachasmimorpha longicaudata were obtained from a>5-yr-old colony at the USDA-CMAVE and reared as described by Sivinski et al.(1996).Parasitoids were maintained in a climate-controlled room with a temperature range of21–24°C and a relative humidity of65–80%. Chemical Analysis Preliminary chromatographic examina-tions of volatile chemicals collected from cut and attached flower were similar.Therefore,due to the handling ease, flowers used for compound identification were cut from the plant stem as close to floral tissue as possible,and the cut end was surrounded with cotton wool soaked in water. Flower heads were placed in a glass volatile collection chamber(Heath and Manukian1992).This system consists of a chamber(30cm long and4cm outside diameter[OD]) that has a sintered glass frit at the upwind end and a joint outlet with a single-port collector base.Humidified charcoal filtered air was pushed into one end of the chambers and over the flower heads.Air was pulled out the other end via a vacuum system.Air exiting the chamber passed through a volatile collection filter that contained50mg of Super-Q®(Refined Technologies,Woodland,TX,USA)to collect and hold any volatiles.Afterwards,collection filters were eluted with three aliquots of100µl methylene dichloride to remove volatile compounds.Extracts of samples were analyzed by capillary gas liquid chromatography(GC)using a Hewlett Packard5890 (Palo Alto,CA,USA)equipped with a cool on-column injector and flame ionization detector.The column,a 30m×0.25mm(i.d.)SE-30capillary column(Alltech Assoc.,New Gloucester,ME,USA),was attached to a 10-m length of0.25-mm(id)deactivated fused silica as a retention gap,which was in turn attached to a10-cm length of0.5mm id deactivated fused silica in the injector. Helium(linear flow velocity18cm/sec)was used as a carrier gas.The oven temperature and injector temperatures were programmed to go from60°C(held for5min)to 200°C at a rate of10°C/min.Mass spectral analysis was accomplished with an HP6890GC equipped with a DB-5 MS®column(30m×0.25mm ID×0.25µm,)linked to an HP5973mass spectrometer.Both electron impact(70eV) and chemical ionization(isobutene reagent gas)spectra were obtained.Helium was the carrier gas,and a splitless injector(injector temperature of240°C,split valve delay of 0.5min)was used.The oven temperature was held at35°C for1min,then programmed to increase at a rate of10°C/min to230°C,which was held for10min.The ion source temperature was230°C.Tentative identifications were made by comparison of fragmentation patterns with patterns available in the NIST-MS library and libraries developed at USDA-CMAVE(Gainesville,FL,USA). Identifications were confirmed by comparison of chromato-graphic retention times and mass spectra of natural compounds with those of commercially available standards analyzed on the same instrument. Electroantennagrams To determine if male and female D. longicaudata had a sensory response to L.maritima volatiles(see results),extracts were analyzed with a GC interfaced to both flame ionization(FID)and electro-antennograph detectors.In this manner,antennal responses were matched with FID signals for compounds eluting from the GC.V olatile extracts were prepared in the manner described above,and1-µl aliquots were analyzed on a Hewlett-Packard(HP)5890Series II gas chromatograph equipped with an HP-5column(30m×0.32mm ID×0.25mm)(Agilent,Palo Alto,CA,USA).The oven temperature was held at40°C for5min,then programmed to increase to10°C/min to220°C and held at this temperature for5min.Helium was used as a carrier gas at a flow rate of2.0ml/min.A humidified air stream was delivered over the antenna is at1ml/min.The antennae were excised by grasping the scape with jeweler’s forceps(No.5,Miltex Instrument Company Inc, Switzerland)at the base,and the antennae were removed from the head of either male or female wasps.The extreme distal and proximal ends of the antennae were held between gold electrodes in conductivity gel(Syntech,Hilversum, The Netherlands).The electroantennal detector(EAD)and FID signals were concurrently recorded with a GC-EAD program(Syntech GC-EAD2000,Hilversum,The Nether-lands),which analyzed the amplified signals on a PC. Flight Tunnel Bioassays To determine the response of D. longicaudata to various odor sources,parasitoids were observed in three identical flight tunnels.These flight tunnels were constructed of clear Plexiglas and measured 128cm long×32×32cm,and were located inside two climate-controlled greenhouses.Natural sunlight illuminat-ed the chambers.Although the greenhouses were climate controlled,extremes of outside temperatures caused the internal temperature to fluctuate between21°and27°C and relative humidity between60%and85%.Airflow and air speed were maintained at0.3to0.4m/s by using a variable speed fan at the downwind end.This was the speed that stimulated the most flight in D.longicaudata in previous investigations(Messing et al.1997).Odors were released into the anterior end of the tunnel.Two separate chambers that contained either an odor source or a blank-air controlJ Chem Ecolwere housed outside of the flight tunnel.Chambers were constructed from114-l all-glass aquaria(31×61×61cm). Lids were constructed of clear Plexiglas and fit tightly to prevent outside air from entering the chambers.Purified air was passed through the sample chambers at0.3m/s regulated with adjustable flow meters(Aalborg Instru-ments,Monsey,NY,USA)and into the flight tunnel.Each chamber was connected to a trap inside the front end of the tunnel.The traps were located midway between the top and bottom of the tunnel,and were constructed from clear cylindrical plastic vials(9×5cm)placed horizontally.Both cylinders had orange colored snap tight lids and had a1.3-cm hole in the center to allow air to exit.This provided an opening for the parasitoids to enter in search of the odor source.Traps,washed initially with a mild detergent,were changed with each new odor source,and the tubing connecting the traps to volatile-source container was washed with detergent after each replication.In all flight tunnel experiments,25presumably mated (sexes held together for3–7d)parasitoids were used per day.All parasitoids had access to water,but food was withheld for24hr before use in experiments to enhance response to potential food sources.Flight tunnels were checked hourly from0900to1700hours.A parasitoid inside a trap was recorded as a positive response,and was removed and returned to the tunnel after counting.Each day,the tubes connecting the odor chambers to the traps were switched to prevent positional effects.Ripe mango fruit,Mangifera indica L.,and the flower-ing plants were tested as odor sources.Mangos are attractive to D.longicaudata in flight tunnel tests(Eben et al.2000)and were used as a positive control.Flowering plants were in full bloom and contained in2-l plastic pots. Each of the three odor sources was tested individually against a blank control of clean odorless air.There were 10replicates for males alone and10for females alone, each replicate lasting a day.In addition,there were10-d-long replicates of25females and25males together with L.maritima as the odor source.This was done to ensure that any sexual dimorphism in response was not due to undetermined environmental factors present during sepa-rate-sex replications.Acetophenone was loaded on11-mm sleeve stopper natural red rubber septa by dissolving the synthetic material at various doses in methylene chloride and filling the large well of the septa with100µl of solution.Septa were air-dried for24hr and stored in a freezer,except during use in the wind tunnels.During flight tunnel tests,septa were placed in glass airtight containers(2l)that served as the odor chambers.All volatile loaded septa were run against blank septa loaded with100µl of methylene chloride and air-dried as a control.To find the dose that elicited the greatest response, various concentrations were exposed to parasitoids.An initial dose of1mg did not produce a response,and increasingly lower doses were tested until there was no significant response.Each dose was initially run for3d in sequential decreasing order from highest to lowest.Only females were in the wind tunnel.Acetophenone doses used were as follows:1mg,100µg,50µg,10µg,1µg,100ng, 50ng,25ng,10ng,1ng.If any response was seen,then that volatile dose was run for10d with females and10d with males.Statistical Analyses Statistical analyses were conducted by using SAS programming(SAS Institute Inc.2001).Poly-nomial regression analysis(PROC GLM)was used to examine flight responses over time and the interactions of “time of day×volatile source”on response.Analysis of variance(PROC ANOV A)followed by means separation through the Waller test was employed to compare the mean responses to various doses.Note that the protocol for all wind tunnel experiments included the return of captured females to the population inside the tunnel.While this was a simple means of controlling for insect density and environmental history,the summed captures could include multiple responses by a particular insect.This led us,when comparing a particular volatile to its control,to employ the most conservative method available,the nonparametric “paired sign test”(Zar1974)where a consistently greater response over10replicates to a particular source yields a P value of0.01.Mean and variance data should be interpreted in terms of“responses”and not“insects responding”. ResultsVolatile Identification Gas chromatographic analysis of volatiles from the flowers of each plant revealed the presence of surprisingly few compounds(Fig.1a).After subtraction of compounds present in volatiles from both species,only three were unique to volatiles collected from L.maritima,and only a single compound was common to those collected from intact flowers and from excised flowers.Mass spectral analysis(both electron impact(EI) spectra and chemical ionization(CI)spectra)of the unique compound indicated a mass of120amu with significant losses of15(CH3),28(CH2O).Tentative identifications were made by comparison of fragmentation patterns with patterns available in the NIST-MS library and libraries developed at USDA-CMA VE(Fig.1a).Acetophenone(Fig. 1b)(8.96min;C8H8O,molecular weight120.15g/mol [US EPA,1987])showed a large peak in L.maritima volatiles and was not present in those of S.verticillata.J Chem EcolIdentification was confirmed by comparison of GC reten-tion times and MS of natural compounds with those of commercially available standards analyzed on the same instruments (Fig.1c).Electroantennagrams Both male and female D.longicau-data registered a neuronal response that correlated to the putative acetophenone peak present in L.maritima volatile extracts (Fig.2).Of the three females tested,their averageresponses over three exposures to volatiles were areas (mV/time)of 1.2,0.7,and 1.5.Of the three males tested,their average responses over three exposures were 0.4,0.6,and 1.1.Flight Tunnel Bioassays Males showed no significant response to any odor source.In several instances,they were caught in isolation traps,but in general,they exhibited occasional walking and flying behaviors at the rear of the flight tunnel away from the odor source.Females had different responses to various volatiles (F =240.5,df =9,1430,P =0.001),and were more likely to be trapped by some volatiles than by their corresponding controls (F =35.8,df =1,1430,P =0.001).Responses differed over time in a non-linear fashion (F =67.3,df =1,1430,P =0.001)(Fig.3).However,the responses over time were similar among treatments,i.e.,there was no significant interaction between type of volatile and the temporal pattern of response (F =0.68,df =1,1430,P =0.41).Because it was impractical to control the amount of volatiles emitted by fruit and flowers,there was no direct comparison of these non-formulated volatile sources.Both mango and L.maritima significantly elicited female responses,but S.verticillata did not (Fig.4).ThesetestsFig.2Neuronal responses of (a )female and (b )male Diachasmi-morpha longicaudata to the acetophenone present in volatile extracts of Lobularia maritima flowers.The darker line represents the presence of acetophenone as revealed by gas chromatography and the lighter line the response of neurons to the same volatilesampleFig.1(a )GC analysis of chemical constituents of two flowering plant,Lobularia maritima and Spermacoce verticillata ,volatiles as revealed by mass spectra.The arrow indicates the unique presence of acetophenone in L.maritima.(b )Acetophenone molecule.(c )A mass spectroscopy comparison between the putative acetophenone collected from L.maritima and a known synthetic standardJ Chem Ecolwere followed by exposures of parasitoids to formulated doses of acetophenone,the compound present in L.maritima ,but absent in S.verticillata .Of the 10doses tested,four elicited a response from parasitoids (Fig.4).Acetophenone concentrations of 100,50,25,and 10ng all showed significant attraction,with 25-and 50-ng doses being the most attractive (F =3889,df =3,P =0.003;Fig.5).Acetophenone doses of 1mg,100,50,10,and 1µg,and 1ng elicited no response in female parasitoids in the wind tunnel.When both males and females were simultaneously exposed in the wind tunnel to L.maritima flower volatiles,no males entered the isolation chambers,but female response was similar to the single-sex experiment with L.maritima (mean of 0.9[SE 0.07]responses/hourly obser-vation period;paired sign test,greater response to volatile source equal to 10of 10;P =0.01).DiscussionThese experiments provide additional evidence that not all flowers are equally attractive to any one particular species of parasitoid (Jervis et al.1993).In the field,L.maritima caught more Braconidae,specifically unidentified Opiinae,than did S.verticillata (Rohrig 2006).This difference was substantiated in flight tunnel tests that found a significant flight response by the opiine D.longicaudata to L.maritima but not to S.verticillata .In addition to this corroborative evidence in support of the generality that there is variance in the diversity of parasitoids attracted to flowers,there were several novel/unusual findings.These included:(1)the suggestion that there may be broad phylogenetic preferences for certain flowers,i.e.,the common response of at least two allopatric opiines to the pair of flower species (Rohrig 2006);(2)the identification of a compound from a flower volatile that is attractive to a hymenopteran parasitoid;and (3)the sexual dimorphic response to L.maritima ,with only females attracted to either the complete flower volatiles or the isolated compound unique to L.maritima .Direct comparisons of parasitoid olfactory response to different live flowers can lead to ambiguous results.Although the sizes of the plants used and the overall area of flowers present were similar (Rohrig 2006),the amounts of volatiles released may have been different.Even within a species,odor concentration can vary among flower parts and change diurnally as well as seasonally (Bergstrom et al.1995).The identification of the attractive compounds present in floral odors can help overcome this obstacle.Once isolated,compounds can be presented in fixed amounts,thereby reducing variation in the odors encoun-tered by the test insects.Thus,the identification ofFemale response to odor source-40-20020406080100120o d o r s o u r c ett t mangomean parasitoids capturedFig.4Mean (SE)of summed captures/replicate of females Diachasmimorpha longicaudata exposed to various volatiles and their corresponding blank air controlsFig.3Mean (SE)windtunnel captures of female Diachasmimorpha longicaudata over time in response to flowering Lobularia maritimaJ Chem Ecolacetophenone as a compound both unique to L.maritima and attractive to D.longicaudata supports the difference in attractiveness observed between the two whole plants.The isolation of acetophenone also allowed estimation of optimal release rates.Diachasmimorpha longicaudata had a sexually dimor-phic response to all volatiles that elicited flight;male parasitoids were not attracted to any.This was true when the sexes were tested both separately and together,so that unnoticed environmental cues present during single-sex experiments cannot be responsible for the sexual difference.Males did exhibit oriented flight and entered the isolation traps in response to virgin females +fruit +honey solutions in experiments conducted in the same wind tunnels (C.S.,unpublished data).Thus,the lack of response in the present experiments was due to the volatiles used and not because of male inability to perform in the flight tunnel environment.Messing and Jang (1992)had previously found that female D.longicaudata responded to various host fruit stimuli to a greater extent than did males.Similar sexually dimorphic responses to plant materials are not restricted to hymenopteran parasitoids.For instance,females of the tachinid Eucelatoria bryani Sabrosky are attracted to many more plant volatiles,including those of flowers,than are males (Martin et al.1990).There are several possible explanations of why female,and not male D.longicaudata ,are attracted to acetophe-none.For instance,females may have unique nutritional needs that flower nectar provides.Acetophenone possess a flowery smell and is used in the perfume industry in fragrances such as vanilla,honeysuckle,and jasmine (USEPA 1987).It is emitted as a volatile by several flowers including Centaurea scabiosa L.,a species frequently visited by butterflies (Andersson et al.2002),Elsholtziaargyi Dong (Peng and Yang 2005),and Calanthe sieboldi Decne (Awano et al.1997).In the laboratory,D.long-icaudata feeds avidly on juices of fruits that would be commonly encountered while searching fallen fruit for hosts (Sivinski et al.2006).Ground-foraging female para-sitoids,however,would also be in the general vicinity of flowers and could visit them to obtain additional other nutrients (Koptur 2005).The relatively ambiguous labora-tory evidence that flowers are an important adult food sources for D.longicaudata does not offer strong support for this conclusion (Sivinski et al.2006).Maximum longevities were significantly extended in the presence of flowers (including the presently examined species),but there was no difference between the mean longevities of parasitoids provided with flowers and those given only water.Females might also mistake acetophenone for a host-fruit volatile or a male-produced pheromone.It has been isolated from Rambutan fruit (Nephelium lappaceum L.;Ong et al.1998)and guava pulp (Psidium guajava L.)(Idstein and Scherier 1985),and is also produced by a number of diverse insects as a semiochemical (Kohnle et al.1987;Schulz et al.1993;Aldrich et al.1995;Birkett et al.2004).The latter hypothesis likely can be discarded since neither acetophenone nor closely related compounds are present in the male-produced sex pheromone (Nancy Epsky,unpublished data).However,female D.longicaudata might mistake the flower volatile for a fruit cue.Tephritids use odor to locate fruit,and the same ability would be useful to fruit fly parasitoids (Eben et al.2000).Males of both fruit flies and their natural enemies may not have been subject to the same selection pressures as females to develop sensitivity to fruit odor stimuli.For example,when 4-ethyl-acetophenone in the peel of navel oranges (Citrus sinensis Osbeck)was used as an odor source in electroantennogram studies of Ceratitis capitata (Weid.),the Mediterranean fruit fly,it elicited a significant voltage spike in females,but not in males (Hernandez et al.1996).Levinson et al.(2003)found that female C.capitata sensilla were significantly more respon-sive to orange odor than male sensilla.Electroantennagrams showed that male D.longicaudata sensed acetophenone,but it did not elicit a behavioral response.Regardless of the significance of the acetophenone response,it may lend itself to improved traps and ultimately better control procedures.There are no efficient synthetic lures and traps available to monitor opiine parasitoid populations in the field (Messing 1992).At present,most monitoring and delineation of fruit fly parasitoids is accomplished through laborious and time-consuming fruit sampling (Montoya et al.2000;Sivinski et al.2000).An improved trapping system would prove valuable in several ways.It could be used to determine the presenceofFig.5Mean (SE)captures of female Diachasmimorpha longicaudata exposed to various doses of acetophenone.The regression line fits only those doses that elicited a significant response.Means that share a letter are not significantly differentJ Chem Ecol。