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Biological Control 44 (2008) 79–89

Psyttalia lounsburyi (Hymenoptera: Braconidae), potentialbiological control agent for the olive fruit fly in California

Kent M. Daane a,*, Karen R. Sime a, Xin-geng Wang b, Hannah Nadel a,b,Marshall W. Johnson b, Vaughn M. Walton a,1, Alan Kirk c, Charles H. Pickett d

a Department of Environmental Science, Policy, and Management, University of California, Berkeley, CA 94720-3114, USAb Department of Entomology, University of California, Riverside, CA 92521, USA

c USDA – Agriculture Research Service, European Biological Control Laboratory, Montferrier sur Lez, 34988 St. Gely Cedex, Franced Biological Control Program, California Department of Food and Agriculture, 3288 Meadowview Road, Sacramento, CA 95832, USA

Received 3 May 2007; accepted 30 August 2007Available online 8 September 2007

Abstract

The African parasitoid Psyttalia lounsburyi (Silvestri) was evaluated as part of a classical biological control program directed at theolive fruit fly, Bactrocera oleae (Rossi), in California, USA. Experimental assessment using three non-target species provided some evi-dence that P. lounsburyi restricts its host use to B. oleae. Female P. lounsburyi preferentially searched olives infested with mature third-instar B. oleae, over other non-target plants, but most offspring were reared from olives containing younger (second through young thirdinstar) B. oleae larvae. Developmental time (egg to adult) and adult longevity were significantly affected by temperature and sex, withmales tending to develop faster and females living longer, especially in the lower ranges of temperatures tested. The mean longevityof adult female P. lounsburyi was greatest when honey was available and lowest when they were provided water alone or nothing.The presence of hosts significantly decreased longevity. Females produced an average of 10.2 ± 2.6 progeny during their lifetimes, whichwas lower than expected for a parasitoid adapted to B. oleae and may be a consequence of increased fruit size—the result of cultivationand selection—reducing parasitoid effectiveness on cultivated vs. wild fruit, as well as constraints on oviposition behavior imposed byexperimental design. The results are discussed with respect to the use of P. lounsburyi as a biological control agent for olive fruit flyin California.Published by Elsevier Inc.

Keywords: Bactrocera oleae; Olea; Psyttalia lounsburyi; Biological control; Non-target assessment; Parasitoid biology

1. Introduction

The African parasitoid Psyttalia lounsburyi (Silvestri)(Hymenoptera: Braconidae) was imported to Californiaas part of a biological control program directed at the olivefruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), arecent arrival in the state (Collier and van Steenwyk, 2003;Rice et al., 2003). The literature suggests that P. lounsburyimay be an effective B. oleae parasitoid. In Kenya, it is the

1049-9644/$ - see front matter Published by Elsevier Inc.

doi:10.1016/j.biocontrol.2007.08.010

* Corresponding author. Fax: +1 559 646 6593.E-mail address: daane@uckac.edu (K.M. Daane).

1 Present address: Department of Horticulture, Oregon State University,Corvallis, OR 97331-7304, USA.

most commonly reared parasitoid of B. oleae in wild olives(Copeland et al., 2004). In South Africa, the minor impactof B. oleae in wild olives is largely attributed to the actionof the resident natural enemy fauna (Hancock, 1989), ofwhich Utetes africanus (Szepligeti) is the dominant parasit-oid and P. lounsburyi is a significant component (Walton,2005). Despite its promise, little is known of the biologyof P. lounsburyi. It was identified nearly 100 years ago (Sil-vestri, 1914), but little effort has been made to use it in thelongstanding biological control programs for B. oleae inEurope (Greathead, 1976). These programs have focusedalmost exclusively on Psyttalia concolor (Szepligeti) sincethe 1950s, when an efficient mass-rearing technique for thisparasitoid was developed that uses the Mediterranean fruit

80 K.M. Daane et al. / Biological Control 44 (2008) 79–89

fly, Ceratitis capitata (Wiedemann) (Tephritidae), in artifi-cial diet (Clausen, 1978; Greathead, 1976).

Among the various parasitoids considered for B. oleae

biological control in California (Sime et al., 2006a,b,c),P. lounsburyi is especially attractive because there isevidence, again from the literature, that it is more of aspecialist on B. oleae than other parasitoid species rearedfrom B. oleae. Its known geographic range—collectionshave been made from Kenya and South Africa—is entirelycontained within that of B. oleae (Nardi et al., 2005; Whar-ton and Gilstrap, 1983). Unlike other common braconidparasitoids of B. oleae, such as P. concolor and Bracon celer

Szepligeti, which are often collected from Ceratitis species,in field collections P. lounsburyi has been reared only fromB. oleae (Narayanan and Chawla, 1962; Neuenschwander,1982; Wharton and Gilstrap, 1983; Wharton et al., 2000).The absence of other host records is not for lack of inves-tigative effort. Likely alternative hosts have been rearedthroughout Africa during decades of foreign explorationfor natural enemies of various tephritid pests (Clausen,1978; Clausen et al., 1965; Wharton, 1989; Copelandet al., 2004). That P. lounsburyi has not been reared fromC. capitata despite intensive efforts to find natural enemiesof this pest (Wharton et al., 2000) and despite the fact thatit can be cultured on C. capitata in the laboratory (Billahet al., 2005) suggests that other tephritid species are accept-able but not attacked. Many tephritid parasitoids use cuesfrom host plants to help locate their insect hosts (Messingand Jang, 1992; Godfray, 1994). Therefore, the apparentfield specialization of P. lounsburyi on B. oleae might alsoinclude cues from the olive tree or fruit.

Field evidence of specialization and efficacy alone is notconsidered sufficient for obtaining permission to releaseparasitoids exotic to the USA and often additional experi-mental evidence of host specificity is also required (Hoel-mer and Kirk, 2005; van Driesche and Reardon, 2004).Over 140 native tephritids inhabit California, includingsome endemic species (Foote et al. 1993). In Californiathere is also concern that introduced parasitoids of tephri-tid pests might attack beneficial tephritids used or consid-ered for biological control of weeds. Direct testing ofimported parasitoids’ responses to those tephritids is neces-sary. Currently these include Chaetorellia succinea (Costa),which feeds in the flower heads of yellow starthistle, Cen-

taurea soltitialis L. (Asteraceae) (Balciunas and Villegas,2001), and Parafreutreta regalis (Munro), which formsstem galls in Cape ivy, Delairea odorata Lemaire (Astera-ceae) (Balciunas and Smith, 2006). These weeds can occurnear olive trees, and this may promote encounters with par-asitoids of B. oleae. Yellow starthistle grows in low-eleva-tion disturbed areas, and Cape ivy is widespread incoastal habitats of California.

We conducted a series of experiments to determine thepotential of P. lounsburyi as a biological control agentfor B. oleae in California and to proceed, if warranted, withits field release. First, we evaluated the non-target impactof P. lounsburyi on C. succinea and P. regalis, the two

tephritid biological control agents, and a native species,Rhagoletis fausta (Osten Sacken), which feeds on wild bit-ter cherry fruit (Rosaceae). These non-target species repre-sent the three major feeding niches of native Californiatephritid larvae (flower heads of Asteraceae, galls, andfruits) (Foote et al., 1993). Given the challenges posed bythe large number of tephritid species in California, ourapproach to assess the potential non-target host range ofcandidate parasitoids was to test a small number of tephri-tid species selected to span a broad range of phylogeneticdiversity and larval habitats, and to compare parasitoidbehavioral responses and reproductive success relative tothe target species and its habitat. Second, we determinedbasic biological parameters of P. lounsburyi, includingadult longevity and fecundity, in order to compare itspotential with other braconids imported and evaluatedfor B. oleae biological control. The accumulated experiencegained during these studies will be helpful in developingfield-release protocols and techniques for rearing P. louns-

buryi on B. oleae.

2. Materials and methods

2.1. Sources of insects and plants, and colony maintenance

Separate colonies of B. oleae and P. lounsburyi wereestablished at two University of California facilities, theCollege of Natural Resource’s Quarantine in Berkeleyand the Kearney Agricultural Center’s Insectary in Parlier,California. Bactrocera oleae were reared on olive fruitusing methods described by Sime et al. (2006b). In brief,adult flies were held in ventilated cages (�50 cm3) that wereprovisioned ad libitum with water and a 2:1 mixture ofhoney and dry yeast extract (Fisher Biotech, Fairlawn,NJ). Susceptible olives were exposed to the fly colony untileach fruit had 3–5 oviposition marks, typically <1 day, andthen removed to a separate rearing cage. The inoculatedolives were held until the mature fly larvae exited the fruitto pupate, upon which the puparia were collected andtransferred to Petri dishes. The olives were collected fromorchards in Fresno County. Small to medium-size olives(‘Mission’ or ‘Manzanillo’ cv.) were used for all experi-ments, while olives of various cultivars, including ‘Ascolano’and ‘Sevillano,’ were also used for maintenance of the flyand parasitoid colonies.

The P. lounsburyi colony used in the Berkeley Quaran-tine originated from adults that had emerged from B. oleaecollected in wild (Olea europaea L. subsp. cuspidata (Wall.ex G. Don)) and cultivated (ornamental) olives near Stel-lenbosch, South Africa, and were sent as puparia to theBerkeley Quarantine in June 2004 and August 2005. Aftera USDA-APHIS permit was granted to move P. lounsburyi

out of Quarantine, experiments were conducted at theKearney Agricultural Center using a P. lounsburyi colonyestablished from �400 parasitoids shipped from theUSDA-ARS European Biological Control Laboratory inMontferrier, France, in July 2006. This colony originated

K.M. Daane et al. / Biological Control 44 (2008) 79–89 81

with parasitized B. oleae collected in wild olives in theBurguret Forest, Kenya, in 2005. The colony was initiallymaintained on B. oleae in olives, but starting in May2005 the parasitoids were reared on C. capitata in artificialdiet. These P. lounsburyi were reared on this medium forapproximately 12 generations prior to arrival in California,at which time they were switched to rearing on B. oleae asdescribed above.

Adult parasitoids of both P. lounsburyi colonies wereplaced in ventilated cages (�50 cm3) that were freely provi-sioned with fly-infested olives, water, and a honey–watersolution (50% by volume). Olives were infested with a mix-ture of second and third instar B. oleae, as other Psyttalia

species oviposit into these stages (Mohamed et al., 2003;Sime et al., 2006b). Inoculated olives were then transferredto clean ventilated containers. Mature B. oleae larvaeemerged from the fruit and dropped onto the bottom ofthe container to pupate. These pupae were collected andtransferred to Petri dishes. Unless stated otherwise, allexperiments in Quarantine and at the Kearney AgriculturalCenter took place in climate-controlled rooms (22 ± 2 �C,40% RH, 16:8 L:D supplemented by natural daylight) withwater and honey–water freely provided.

A P. regalis colony, used for non-target assessment, wasmaintained at Berkeley Quarantine in summer and fall2004. The colony was established using stock (originallyfrom South Africa) maintained at the USDA—ARS quar-antine facility in Albany, California. Rearing methods usedare described by Balciunas and Smith (2006). In brief, pot-ted Cape ivy plants were placed in 32 · 45 · 96 cm sleevecages with adult P. regalis for 4–7 days and then removedto racks under filtered daylight for gall maturation. Maturegalls were cut from the plants with a few cm of stem, andplaced in moist blocks of Oasis� floral foam (Smithers-Oasis, Cuyahoga Falls, OH) for adult fly emergence.

A second non-target tephritid species, C. succinea, wasmaintained using rearing methods based on thosedescribed by Balciunas and Villegas (2001). Adult C. succi-nea were reared from yellow starthistle flowers collected inContra Costa and Yolo counties from July through Octo-ber 2004. The emerging adult flies were caged and suppliedwith water and a mixture of dry yeast extract and honey.To produce flower heads infested with groups of same-agedlarvae, potted plants were pruned of all but the suitableyoung flower buds, up to a week before expected anthesis,and then placed in a cage with the reared adult flies (6- to14-day-old) for 2 days. The plants were then moved to agreenhouse (23 ± 3 �C) to rear the larvae, which were usedin non-target studies when they reached the appropriatestage.

The third non-target species, the native black cherryfruit fly, R. fausta, is a univoltine species found in ripeningbitter cherry, Prunus emarginata (Douglas ex Hooker)Eaton, in late summer and fall. Branches bearing fruit withevidence of R. fausta infestation (oviposition scars) werecollected on the western slope of the Sierra Nevada moun-tains (Fresno County) in August and September 2004 and

brought directly to the Berkeley Quarantine forexperiments.

2.2. Assessment of host range

Non-target tests were conducted in Quarantine using theSouth African P. lounsburyi. All experiments were con-ducted in small cages (25 cm3) with glass fronts and screensides. Each test consisted of two phases: a no-choice phasein which parasitoids were offered only the non-target mate-rial, and a positive-control phase in which olives infestedwith B. oleae were added to the cages to confirm thatP. lounsburyi were capable of oviposition into B. oleae.All plant materials were then incubated for at least 6 weeksto allow adult parasitoid or fly emergence. If no adultsemerged, the plant material was dissected and fly pupariawere examined to determine if they had been parasitized.Additional cages were set up as negative controls, in whichnon-target hosts and B. oleae were set up in parallel withthe no-choice tests and positive controls, and parasitoidswere excluded from the treatment.

The non-target fly species (C. succinea in the flower budsof yellow starthistle, P. regalis in self-formed galls in Capeivy, and R. fausta in cherry fruit) were chosen in part tomatch B. oleae in size and because the galls, fruit, and budsare comparable in size to olive fruit. Adequate host sizewas considered important to avoid spurious parasitoidmortality due to insufficient host resources, while the sizeof the infested structures was important to minimize nega-tive results due to hosts that were buried beyond the reachof parasitoid ovipositors. Most tephritid larvae hadreached the third instar when the experiments were initi-ated. All were presented in situ (i.e., in the gall, fruit, orbud) in bouquets 10–20 cm in length, with the base in waterand leaves removed to allow a clear view of wasp behav-iors. Buds of yellow starthistle were exposed to adultC. succinea 10–12 days before use in experiments. Dissec-tion of subsamples revealed that most flower heads wereinfested, and that over 60% of the larvae were in the thirdinstar. Cape ivy galls were harvested 20–27 days after expo-sure to P. regalis. Subsamples of bitter cherries were dis-sected to ensure that the majority of R. fausta reachedthe third instar.

In the first (no-choice) phase of the experiment (first48 h), a bouquet of the non-target plant material wasplaced in each cage. The amount of parasitoid and hostmaterial in each replicate depended on availability of mate-rial and consisted of 6–15 mated female P. lounsburyi,which were previously exposed to B. oleae, with either8–10 galls (P. regalis), 10–20 flower heads (C. succinea),or 20 fruit (R. fausta) in each cage. Each replicate beganbetween 8:00 and 9:30 AM. The second (positive-control)phase began at 9:00 AM on the third day, when 8–10 olives(‘Manzanillo’, ‘Mission’, or ‘Sevillano’ cultivars) infestedwith 5–10 third-instar B. oleae were placed in the cage ata height level with the non-target material. This phaselasted an additional 48 h.

82 K.M. Daane et al. / Biological Control 44 (2008) 79–89

On the first day of both the no-choice and the positive-control phases, the parasitoids were observed for 10 min at10:00, 13:00, and 16:00 h. The number of P. lounsburyi incontact with the galls, flower heads, or fruit was recordedunder three categories: investigating, probing, or incidentalcontact. ‘‘Probing’’ was defined as the insertion orattempted insertion of the ovipositor. ‘‘Investigating’’ wasdefined as a walk with slightly lowered antennae, inter-rupted by frequent stops with the antennae raised. Otherbehaviors on the host material, such as standing motion-less, grooming, or brief contact, were recorded as incidentalcontact.

At the end of the positive-control phase, fruit flies wereseparated and incubated for a minimum of 6 weeks. Bou-quets of galls, still maintained on stems in water, were con-fined in vials with mesh windows, while flowers and fruitwere removed from stems and held in plastic vials (flowers)or paper cups (fruit) with mesh lids. The numbers of adultflies and parasitoids that emerged were recorded. Galls andflower heads were later dissected and the number of deadlarvae, pupae, and adults inside recorded. Fruit were dis-sected only in replicates lacking parasitoid emergence.The negative controls were treated in a similar manner.Unemerged puparia were dissected after the 6 week incuba-tion period to determine if they contained unparasitizedhost pupae or encapsulated eggs or cadavers of parasitoids.Dead fly pupae with recognizable form were assumed to beunparasitized because the Psyttalia species we studiedkilled the host directly after formation of the puparium.Unencapsulated parasitoid eggs and first instars couldnot be detected with this technique. Therefore, hostremains that had clearly not differentiated into pupae andalso lacked later parasitoid instars were treated as ‘‘unde-termined’’ and excluded from the analysis.

2.3. Host stages preferred for oviposition

The preference of P. lounsburyi for olives infested withdifferent B. oleae stages was examined. The experimentwas conducted at the Kearney Agricultural Center (usingKenyan P. lounsburyi). To produce an age series of B.

oleae, fresh olives were exposed to flies for 2 h every 2–3days and then held at 25 ± 1 �C. Olives used had 3–5 ovi-position marks. Immature stages inside the olives werepresented to the parasitoids at 1, 3, 6, 8, and 10 days,using a 2 days exposure period each time, to create fivehost age categories of 1–2, 3–4, 6–7, 8–9, and 10–11 days.A sub-sample of olives from each set was dissected beforeand after each test to determine which host stages werepresent. Under these conditions, 1–2 days post exposureolives contained mostly eggs and a few first instars; 3–4days post exposure olives contained first and secondinstars; 6–7 days post exposure olives contained secondinstars and young third instars; 8–9 days post exposureolives contained mostly third instars; and 10–11 days postexposure olives contained mature third instars and occa-sionally puparia.

At the start, four female P. lounsburyi were placed in aventilated cage (30 cm3) along with a fresh-cut olive branch(in water) for a 1 day acclimation period. Thereafter, fourolives of each age category (20 total) were added for a 2day oviposition period. Olives containing B. oleae hostsof the same age were grouped in a small Petri dish (5-cmdiameter), and the five small Petri dishes were then placedinside a large Petri dish (15 cm diameter). To quantify theparasitoid’s searching activity on different host stages, two30 min observation sessions were made, one in the morningand the other in the afternoon on the first day of exposure.During these sessions, the number of parasitoids observedon olives was recorded at 3 min intervals to estimate therelative amount of parasitoid searching time on differenthost stages. After the exposure period the olives were heldat 25 ± 2 �C to rear either adult parasitoids or flies.

2.4. Pre-imaginal developmental rates at different constant

temperatures

Developmental rates (egg to adult) of P. lounsburyi wereassessed at three constant temperatures (17.9 ± 0.7,23.9 ± 1.1, and 27.1 ± 1.2 �C) in temperature cabinets.Olives infested with second and third instar B. oleae wereexposed to ovipositing P. lounsburyi for 24 h. An over-abundance of B. oleae was presented (about 10 infestedolives per female wasp) to limit the level of superparasitism.After exposure to P. lounsburyi, the infested olives wereapportioned evenly among temperature treatments. A Petridish of water was placed inside each temperature cabinet tomaintain the humidity. The containers were checked twicedaily (morning and afternoon) to record the number andsex of emerging parasitoids. Twenty replicates were madefor each temperature.

2.5. Adult longevity

Adult male and female longevity were measured atseven temperatures (15.2 ± 0.3, 21.9 ± 0.2, 24.8 ± 0.4,27.8 ± 0.2, 30.1 ± 0.2, 32.0 ± 0.5, and 34 ± 0.5 �C). Newlyemerged parasitoids were isolated in glass vials (5-cmlong · 1-cm diameter, with a mesh lid) and randomlyassigned to a temperature treatment. The parasitoids werechecked daily for mortality. At each temperature, 10 maleand 10 female parasitoids were tested.

Adult female longevity was also compared among fivetreatments of different host and/or food provisions: (1)olives containing hosts, honey–water (50% by volume),and water; (2) uninfested olives, honey–water and water;(3) honey–water and water only; (4) water only; and (5)no provisions. Newly emerged females were collected daily,transferred to a small container with males, supplied withwater and honey–water, held for 2 days to mate, individu-ally isolated in ventilated plastic containers (15 cm diame-ter · 6 cm deep), and then randomly assigned to atreatment. Olives (four per container) were replaced everyother day. Where olives with hosts were offered, each olive

Fig. 1. Comparison of mean (±SEM) number of responses of female P.

lounsburyi to non-target (NT) hosts and B. oleae recorded during three 10-m observation periods in a 48-h no-choice phase (only NT) and three 10-mobservation periods during a subsequent 48-h choice phase (NT and B.

oleae). The responses were categorized as: (A) incidental contact withoutany apparent response to hosts; (B) investigation behavior indicatingawareness of host presence; and (C) probing the substrate with theovipositor. The non-target hosts were Chaetorellia succinea in yellowstarthistle flower heads, Parafreutreta regalis in Cape ivy stem galls, andRhagoletis fausta in bitter cherry fruit. Bactrocera oleae were offered inolives. Different letters above each group of bars indicate significantdifferences (one-way ANOVA, followed by Tukey’s HSD, P < 0.05).

K.M. Daane et al. / Biological Control 44 (2008) 79–89 83

had 5–10 fly oviposition marks (20–40 larvae for each 2 dayinterval) and the fly larvae were at a suitable stage (secondand third instars) for parasitoid oviposition. Parasitoidswere checked daily for mortality. Ten replicates were madeper sex per treatment. Experiments were conducted at theBerkeley Quarantine using South African P. lounsburyi.

2.6. Fecundity

Lifetime fecundity was investigated at the Kearney Agri-cultural Center, using the Kenyan P. lounsburyi. Newlyemerged female and two male P. lounsburyi were placedin a ventilated cage (15 · 15 · 20 cm3). Ten infested olives,each containing 3–5 second or third instar B. oleae, werecontinuously provided during the adult parasitoids’ life-time. The olives were presented on a Petri dish (8.5 cmdiam), raised 5 cm above the cage floor. Every 2 days,the exposed olives were replaced with new olives. MaleP. lounsburyi were removed after 4 days. The exposedolives were isolated according to exposure dates, and incu-bated as described previously. Emerging flies or parasitoidswere recorded daily. Four replicates were made of eachtreatment, with 4–9 females in each replicate. A sub-sampleof newly emerged female P. lounsburyi from the colony wasdissected to estimate egg load. All tested female parasitoidswere immediately dissected after death to determine thenumber of mature eggs that were not laid.

2.7. Statistics

Results are presented as means per treatment (±SEM).Treatment effects for host-range and development timewere analyzed using one-way ANOVA, and treatmentmeans were separated using Tukey’s HSD test(alpha = 0.05) when there were three or more treatments(JMP 6.0.3, SAS, Cary, NC). Proportional data were trans-formed (arcsine square-root) before ANOVA. Adult lon-gevity data were analyzed with the Cox survival analysismodel and Kaplan-Meier estimator (Systat 12, Systat Soft-ware, Inc., San Jose, CA). Female and male longevity werecompared using the parametric model function with genderas the strata and temperature as the covariate; the outputprovided the Mantel–Haenszel log-rank test for strata sep-aration (Chi-square) and a Z-test for impact of the covar-iate. Temperature and food provision effects on adultfemale longevity were similarly compared for all possiblepair-wise treatment comparisons, using the nonparametricmodel function. In these analyses, the experiment-wideerror rate used was alpha’ = alpha/n, where alpha = 0.05and n is the number of possible pair-wise comparisons.

3. Results

3.1. Assessment of host range

Psyttalia lounsburyi showed almost no interest in thenon-target hosts (Fig. 1). Incidental contact with the non-

target host material was observed at low rates in all testsbut was not different between the no-choice and choicephases of the studies (Fig. 1A). Neither probing (Fig. 1B)nor investigation behaviors (Fig. 1C) were observed inthe no-choice phase of the experiment, except for two inci-dences of investigation behavior observed in the choicephase on Cape ivy galls. The parasitoids did, however,investigate and probe olives with B. oleae larvae in thechoice phase of the experiment (Fig. 1), and offspring wererecovered from these hosts (Table 1). Psyttalia lounsburyi

investigated olives more often than yellow starthistle(Fig. 1B, one-way ANOVA, F = 7.64, df = 2, 32,P = 0.002; Tukey’s HSD, P < 0.05) and bitter cherry fruit(Fig. 1B, one-way ANOVA, F = 10.00, df = 2, 14,

Table 1Number of parasitized and unparasitized offspring reared from B. oleae and non-target hosts during the P. lounsburyi host-specificity study

Non-targethost

Offspring reared from hosts exposed to parasitoids (no-choice and choicephases)

Offspring reared without exposure to parasitoids (negativecontrol)

Non-target hosts B. oleae Non-target hosts B. oleae

na Flies Parasitized Flies Parasitized n Flies n Flies

C. succinea 11 130 0 473 30 8 96 11 509P. regalis 10 172 0 746 39 5 60 7 664R. fausta 5 21 0 182 29 5 34 5 252

a Here ‘‘n’’ is the number of replicates for each combination of target and non-target studies, ‘‘Flies’’ is the number of adult fruit flies reared, and‘‘Parasitized’’ includes both adult P. lounsburyi and immature cadavers.

Fig. 2. Host-stage preference as (A) mean percentage (±SEM) adultfemale Psyttalia lounsburyi on olives containing hosts of a given agecategory during timed observation intervals (linear regression: y = 4.05x–6.74, r2 = 0.818; F = 13.56, df = 1,4, P = 0.034) and (B) as mean percent-age (±SEM) of P. lounsburyi offspring that emerged from different hostage categories (log normal ‘‘peak’’ regression: y = a exp(�0.5(ln(x/x0)/b)2), where a, b, and x0 are 35.76, 0.51, and 5.34, respectively, F = 37.66,df = 2,4, P = 0.026). Within in graph, different letters near each meanindicate significant differences (one-way ANOVA, P < 0.05).

84 K.M. Daane et al. / Biological Control 44 (2008) 79–89

P = 0.003; Tukey’s HSD, P < 0.05) in both phases of thestudy. When P. regalis in Cape ivy galls was offered with-out choice, the parasitoids investigated the galls less thanthe olives that were offered during the choice phase(Fig. 1B, one-way ANOVA, F = 3.59, df = 2, 29,P = 0.041; Tukey’s HSD, P < 0.05). However, during thechoice phase the difference in P. lounsburyi’s responses togalls or olives was not significant. Throughout the non-tar-get host study, probing was observed only in olives but wasrare even in the target host and was higher only when com-pared with Cape ivy galls (Fig. 1C, one-way ANOVA,F = 6.00, df = 2, 29, P = 0.007; Tukey’s HSD, P < 0.05).

3.2. Host-stage preference and reproductive success

The percentage of adult P. lounsburyi observed on theolives differed across age categories tested (F = 5.35,df = 4, 95, P < 0.001) (Fig. 2A). More parasitoids wereobserved searching olives containing the oldest host larvae,than those containing egg to small third instar fly larvae.Across all age categories, 4.0 ± 0.6 adult P. lounsburyi werereared per replicate. Parasitoids were reared from all hostage categories, but more were reared from the 3- to9-day-old larvae (late 1st instar to 3rd instar) than from1- to 2-day-old larvae (egg to early first instar) (F = 5.18,df = 4, 115, P < 0.001) (Fig. 2B).

3.3. Pre-imaginal developmental time at different constant

temperatures

Developmental time (egg to adult) of P. lounsburyi wassignificantly affected by temperature and sex (temperature:F = 2081.6, df = 2, P < 0.001; sex: F = 14.2, df = 1,P < 0.001; temperature · sex: F = 2.6, df = 2, P = 0.07).Under each temperature the developmental time of femaleP. lounsburyi was significantly longer than that of males.Developmental rates for both male and female waspsincreased with temperature (Fig. 3).

3.4. Adult longevity

Across all temperature treatments, adult females livedlonger than males (Fig. 4A). Temperature treatmentshad a significant impact (covariate analysis: Z = 8.669,

P < 0.001) and for that reason female and male data wereanalyzed separately. Adult longevity ranged from an aver-age 46.8 ± 3.4 days (15 �C) to <1 day (34 �C), generallydecreasing with increased temperature for both male andfemale P. lounsburyi although at 25 �C both longevitieswere lower than expected (Table 2). Survival analysis is

Fig. 3. Mean developmental time (egg to adult) (±SEM) of Psyttalia

lounsburyi at different constant temperatures. Different letters over eachbar within the same sex (female indicated by capital and male by lowercase letters) indicate significant differences (one-way ANOVA, P < 0.05).

Fig. 4. Survivorship curves for adult Psyttalia lounsburyi for (A) male andfemales with data combined across all temperatures showing females arelonger lived than males (Mantel–Haenszel log-rank v2 = 6.496, df = 1,P = 0.011) and (B) female P. lounsburyi at six temperature treatments(Mantel–Haenszel log-rank v2 = 114.5, df = 6, P < 0.001). Different lettersfollowing each strada in the keys indicate a significant difference insurvival analysis pairwise comparisons for gender (alpha = 0.05) andtemperature (alpha = 0.0033).

Table 2Longevity (±SEM) of adult female and male P. lounsburyi when held atconstant temperatures and provisioned with honey and water

Temperature (�C) Adult longevity (in days)

Female Male

15 46.8 ± 3.4 25.7 ± 4.722 22.4 ± 3.8 11.8 ± 2.225 10.7 ± 1.7 5.3 ± 1.028 14.3 ± 1.9 11.2 ± 1.230 11.2 ± 1.7 9.2 ± 0.732 2.4 ± 0.2 2.0 ± 0.234 <1.0 <1.0

K.M. Daane et al. / Biological Control 44 (2008) 79–89 85

presented for adult females only, although males showed asimilar pattern. At 34 �C, no adults survived to the firstobservation period (day 1) and these data were notincluded in the analysis. Adult females lived longer at15 �C and progressively shorter with increasing tempera-tures (Fig. 4B). Longevity was affected by provisioningtreatment, as indicated by survival analysis (Fig. 5). FemaleP. lounsburyi lived longest in the ‘‘honey and water’’(61.8 ± 8.2 days) or ‘‘olives, honey and water’’(67.0 ± 7.9 days) treatments, followed by ‘‘olives infestedwith fly larvae’’ (32.4 ± 1.8 days), then ‘‘water alone’’(11.0 ± 1.7 days), and finally with no provisions(4.7 ± 8.2 days).

3.5. Fecundity

Over its lifetime, P. lounsburyi produced 10.2 ± 2.6 off-spring per female, of which 60.6 ± 5.9% were female. Theten olives used per 2 day exposure period contained a totalof 27.1 ± 1.5 (n = 56) host larvae based on dissection ofrandom samplings of infested olives; therefore mostof the presented hosts were unparasitized. Dissection of

Fig. 5. Adult female Psyttalia lounsburyi longevity given different foodprovisions. Different letters following each provision treatment in the keyindicate a significant difference in survival analysis pairwise comparisons(alpha = 0.005).

Fig. 6. Mean number (±SEM) of Psyttalia lounsburyi offspring producedper parasitoid over her lifetime when provided with unlimited quantifies ofB. oleae larvae.

86 K.M. Daane et al. / Biological Control 44 (2008) 79–89

exposed fruit after all flies and parasitoids had emergedrevealed that percentage of dead puparia was low(2.48 ± 0.47%, n = 27). Thus, the results appear to reflectthe maximum reproductive potential of the parasitoidunder the given laboratory conditions, rather than parasit-ized but undeveloped P. lounsburyi.

Female P. lounsburyi in this experiment survived41.6 ± 3.5 days (range 12–70 days). Newly emerged females(<12 h) contained few mature eggs (1.17 ± 0.56 per female,n = 12). Egg maturation appeared to proceed quickly.Females dissected 24–48 h after emergence and deprivedof hosts contained 17.2 ± 2.1 mature eggs (n = 13). Theparasitoids began to oviposit on the second day after emer-gence and reached peak oviposition tempo at 6–10 days(Fig. 6). Oviposition activity decreased thereafter andnearly stopped after 30 days, although females still con-tained 16.6 ± 3.1 eggs at death. Of the 20 females tested,three did not produce any offspring.

4. Discussion

Results of non-target assessment provide evidence thatP. lounsburyi has a narrow host range or behaviors thatlimit host searching to olives or similar fruit. The experi-mental method used was relatively conservative, as thesmall cage size promoted high encounter rates with theinfested plant material regardless of the type of long-rangeorientation cues used by the parasitoid to locate a particu-lar host-plant complex. The lack of investigation or prob-ing in bitter cherries, the most likely alternate habitattested to induce a searching response from a parasitoidadapted to a frugivorous host, provides support for areduced risk to non-target tephritids by P. lounsburyi. Inconcurrent tests under the same conditions, other braconidolive fly parasitoids, P. concolor and Bracon celer Szepligeti,and braconids that attack other tephritids, Diachasmimor-

pha kraussii (Fullaway), and D. longicaudata (Ashmead),

investigated and probed bitter cherries at high rates(unpublished data). Further evidence for strong olive hab-itat specificity is provided by probing and reproduction ofP. lounsburyi on C. capitata larvae inserted into olive fruit(R. Wharton, personal communication).

Taken with evidence from African field surveys, thesedata argue that P. lounsburyi specializes on B. oleae by vir-tue of either a limited range of acceptable host species orsearching behavior that is limited to olives. Host specificityarises from behavioral and physiological adaptations thatlimit the range of organisms that parasitoids will attackand utilize as hosts, and behavioral adaptation precedesphysiological adaptation in the evolution of host specificity(Futuyma and Moreno, 1988). Host-habitat specificity maybe more advantageous in a biological control agent thanphysiological specificity, as it may serve to focus hostsearching on olives, thereby improving parasitoid efficiencyin addition to reducing potential risk to non-target hosts.For that reason, information developed from laboratorycage studies may not reflect the actual host range. Also,only three non-target tephritids were evaluated. We hadhoped to test more native tephritid species; however, twoyears of field collections for known California native spe-cies recovered insufficient material that coincided with par-asitoid availability, and we were unable to develop coloniesof those that were recovered. Moreover, P. lounsburyi

response to B. oleae was far lower than that recorded forother braconid species tested in Berkeley Quarantine (Simeet al., 2006b,c), suggesting that experimental conditionswere not ideal for normal behavior. Several authors haveemphasized the importance of in situ parasitoid behaviorsand host microhabitat in non-target evaluations (Messing,2001). For example, Duan and Messing (1997) suggest thatthe spheroid shape of a non-target host substrate, such asgalls, may be similar enough to small fruit to elicit host-searching behaviour in opiine fruit fly parasitoids whileflowerheads of Asteraceae shrubs housing flowerheadtephritids have no resemblance to the host fruits of frugiv-orous tephritids.

Studies of P. lounsburyi biological parameters providesome important information for field release and insectaryproduction guidelines. Although P. lounsburyi has beensuccessfully reared from B. oleae attacked in the early thirdinstar (Billah et al., 2005), its preference for different hoststages and relative success in parasitizing them have notbeen investigated, nor has its behavior been described whenoffered B. oleae feeding in fruit. Female P. lounsburyi showa preference for older third-instar host larvae, comparedwith egg to second instar stages (Fig. 2A). However, therewas a poor correlation between the host stages attackedand host stages producing the most offspring (Fig. 2B).This discrepancy may be explained as the outcome of twoindependent factors: an increased ability to locate older lar-vae in the fruit, but a decreased ability to oviposit in olderlarvae in cultivated olives. Many parasitoids locate hiddenhosts through substrate vibrations (Glas and Vet, 1983;Van Dijken and Van Alphen, 1998), and strong evidence

K.M. Daane et al. / Biological Control 44 (2008) 79–89 87

exists that a congener of P. lounsburyi, P. concolor, locateshosts through vibrotaxis (Canale and Loni, 2006). Thesuites of searching behaviors exhibited by P. concolor andP. lounsburyi on infested olives are the same (H. Nadel,personal observation). Psyttalia concolor responds morestrongly to the third instar than the second instar of C. cap-itata in experimental arenas (without fruit); presumablybecause the larger instar produces stronger or more fre-quent vibrations while feeding (Canale and Loni, 2006).Psyttalia concolor does not reject either of these instarsonce it locates them, and also develops with equal successin both (Canale and Loni, 2006). For P. lounsburyi, thethird instar induces more searching behavior, but the thirdinstar B. oleae feed deeper than the second instar in culti-vated olives, which are much fleshier than wild olives.For most of the third instar, therefore, the larvae arebeyond the reach of the short P. lounsburyi ovipositor(<2 mm) (H. Nadel, unpublished data).

The results of our P. lounsburyi host-age preferencestudy show a clear discrepancy between the age categorieson which adult parasitoids most commonly searched andprobed (Fig. 2A) and from which parasitoids were reared(Fig. 2B). These data lend further support to our hypothe-sis that the shorter ovipositor of P. lounsburyi cannot reachthe larger host larvae that feed deeper inside the cultivatedolive—although these host stages may be acceptable andeven preferred. It is also possible that the older larvae areparasitized but better able to encapsulate the parasitoidegg. These results differ from those found for other Psyttal-

ia species that were tested similarly using an age series of B.oleae larvae. Psyttalia ponerophaga (Silvestri) showed nodiscrepancy between preference and reproductive success,but rather an increase in both with larval age (Simeet al., 2007). A discrepancy was observed in P. concolor

(Sime et al., 2006b), but it is different than that found inP. lounsburyi, and also differs from the predictions ofCanale and Loni (2006). In Sime et al. (2006b), P. concolor

preferred to oviposit into second and young third instars,but most larvae were reared from hosts attacked as thirdinstars. Here, the discrepancy was proposed to result fromthe relatively short ovipositor of Psyttalia species and thepeculiar pre-pupation behavior of B. oleae (Sime et al.,2006b, 2007). In the pulp of cultivated olives, which is typ-ically much deeper than the length of a Psyttalia oviposi-tor, third-instar B. oleae generally feed closest to the pit,but shortly before pupation the older third instar tunnelsnear the surface to create an exit ‘‘window’’. The late thirdinstar may, therefore, be the most vulnerable to attack bysome Psyttalia species.

Temperature tolerances will also be an important factorin the establishment and performance of parasitoids in Cal-ifornia because B. oleae infestations are found in both therelatively cool coastal and hot inland areas. In our investi-gation of pre-imaginal developmental rates, a relativelyhigh mortality of parasitized hosts (70–80%) was observedat the highest temperature (27 �C), which suggests that P.

lounsburyi has a relatively low upper threshold for develop-

ment. These results do not bode well for P. lounsburyi

establishment in the inland valleys. However, because thesedata were collected using hosts feeding on picked fruit, theymay not reflect performance in the field. Collected olivestend to dry out and shrivel when held at temperaturesabove 25 �C, which may impede parasitoid development(Sime et al., 2007). Fruit still on a tree would remain turgidat even higher temperatures. Therefore, the temperaturedevelopment rates are better used to compare P. lounsburyi

to other parasitoid species tested against B. oleae using thesame experimental conditions. The developmental ratesand mortality of P. lounsburyi are similar to those observedfor other B. oleae parasitoids tested, including D. kraussii

and D. longicaudata (Sime et al., 2006c), P. ponerophaga(Sime et al., 2007), and P. concolor (Sime et al., 2006b).This finding indicates that P. lounsburyi would performas favorably as other species being considered for use inCalifornia, based on temperature tolerances, across therange of climates found in the olive-growing regions.

Similarly, overall patterns of adult longevity are compa-rable to these other species, apart from P. concolor, whichcan live for long periods relative to the other species at tem-peratures of 30 �C or higher (Sime et al., 2006b). Withrespect to provisioning, the decrease in adult longevitywhen host-infested fruit were available suggests that theparasitoids expended energy in attempting or succeedingto parasitize hosts. This suggests that the parasitoids wereable to distinguish host-infested olives from those lackinghosts. Identical patterns were observed in P. concolor (Simeet al., 2006b), D. kraussii, and D. longicaudata (Sime et al.,2006c), though not in P. ponerophaga, which respondedequally to all three treatments in which honey was provided(Sime et al., 2007). The argument could also be made that,without hosts, these Psyttalia species resorb eggs to utilizereserves (Greathead 1976).

In contrast, P. lounsburyi fecundity was lower than otherspecies tested as B. oleae parasitoids under similar experi-mental conditions, including P. ponerophaga (18.7 ± 2.9offspring per female) (Sime et al., 2007), two laboratorystrains of P. concolor (22.5 ± 5.1 and 28.7 ± 4.1 offspringper female) (Sime et al., 2006b), and two Diachasmimorpha

species (23.6 ± 5.3 and 22.7 ± 5.5 offspring per female)(Sime et al., 2006c). This result is somewhat surprisingbecause we expected P. lounsburyi, as an adapted specialist,to perform better on B. oleae feeding in fruit comparedwith the more polyphagous P. concolor or the two Diacha-smimorpha species for which B. oleae is an entirely novelhost. One hypothesis to explain low parasitism rates againconcerns the short ovipositor of P. lounsburyi relative tothe feeding depth of hosts within cultivated olives. Wildfruit are small, around 7–8 mm in diameter, while culti-vated varieties are typically 2–3 cm in diameter (Bartoliniand Petruccelli, 2002; Tzanakakis, 2003). In the field, ithas been reported that P. lounsburyi is not found as fre-quently in cultivated olives as in wild olives. A survey inSouth Africa produced 22 individuals from wild olivesand one from cultivated olives (Neuenschwander, 1982).

88 K.M. Daane et al. / Biological Control 44 (2008) 79–89

In contrast, the same survey found that B. celer, which hasa longer ovipositor (�3 mm, instead of <2 mm) and favorsmature larvae, was about equally abundant in wild and cul-tivated olives. The hypothesis is also supported by theresults of the longevity experiment comparing responsesto different provisioning regimes.

The poor reproductive performance of P. lounsburyi

compared with the other Psyttalia species indicates thatthere are confounding factors in addition to the shortnessof its ovipositor. One alternative explanation for ourresults compared with the field observations hinges onthe chemical cues used to orient to and identify host larvaeand the host/plant complex. Domestic olives differ chemi-cally from wild olives (Massei and Hartley, 2000). Thesedifferences could disrupt the parasitoid’s host-searching,host-identification, or ovipositional behaviors, or impedelarval development. Other differences in chemistry arelikely between picked olives, as used in these experiments,and fruit still on the tree. The more specialized P. louns-

buryi is expected to be especially sensitive to changes inplant chemistry, and to have innate, relatively inflexibleresponses to semiochemicals used to find hosts and plants(Vet and Dicke, 1992).

Previously, no effective rearing method had been devel-oped for P. lounsburyi, which no doubt contributed to thelack of interest in this species. In California, rearing on C.

capitata is not an option, because the Mediterranean fruitfly is not established in the state and importation to thecontinental USA is currently forbidden (Headrick andGoeden, 1996). Another drawback of this rearing methodis that it may select for behaviors that are disadvantageousin the field (Kimani-Njogu et al., 2001). The methodsdescribed here can be used to rear P. lounsburyi year round;however, costs are high and production is low. The use ofartificial diet is a plausible option and is currently beinginvestigated to mass produce P. lounsburyi during periodswhen susceptible olives are not available.

Acknowledgments

John Andrews oversaw importation and rearing opera-tions at Berkeley I&Q. Rob Stotter (University of Stel-lenbosch) collected and shipped parasitoids from SouthAfrica. Kim Hoelmer, Mike Pitcairn, and Walker Jonesprovided advice and assistance at various stages of the pro-ject. Nadia Tomic-Carruthers helped initiate the C. succi-

nea rearing system, and Jim Brown (CDFA) providedpotted thistle and ivy plants. Cape ivy flies were providedby Joe Balciunas (USDA-ARS). We also thank H. Beeson,M. Gerik, L. Miljkovic, S. Mortezaei, M. Orsini, K. Rear-don, and M. Yamamoto for help with laboratory experi-ments. Funds were provided by the California SpecialtyCrop Block Grant (administered by the California Depart-ment of Food and Agriculture, with funding from the Uni-ted States Department of Agriculture) and the CaliforniaOlive Committee. Voucher specimens are deposited inBerkeley I&Q.

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