Psyttalia Lounsburyi (Hymenoptera: Braconidae), Potential Biological Control Agent for the Olive Fruit Fly in California

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Biological Control 44 (2008) 79–89 www.elsevier.com/locate/ybcon

Psyttalia lounsburyi (Hymenoptera: Braconidae), potential biological 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, USA b Department of Entomology, University of California, Riverside, CA 92521, USA c USDA – Agriculture Research Service, European Biological Control Laboratory, Montferrier sur Lez, 34988 St. Ge´ly Cedex, France d Biological Control Program, California Department of Food and Agriculture, 3288 Meadowview Road, Sacramento, CA 95832, USA

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

Abstract

The African parasitoid Psyttalia lounsburyi (Silvestri) was evaluated as part of a classical biological control program directed at the olive fruit fly, Bactrocera oleae (Rossi), in California, USA. Experimental assessment using three non-target species provided some evidence 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 third instar) B. oleae larvae. Developmental time (egg to adult) and adult longevity were significantly affected by temperature and sex, with males tending to develop faster and females living longer, especially in the lower ranges of temperatures tested. The mean longevity of 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, which was lower than expected for a parasitoid adapted to B. oleae and may be a consequence of increased fruit size—the result of cultivation and selection—reducing parasitoid effectiveness on cultivated vs. wild fruit, as well as constraints on oviposition behavior imposed by experimental design. The results are discussed with respect to the use of P. lounsburyi as a biological control agent for olive fruit fly in California. Published by Elsevier Inc.

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

1. Introduction most commonly reared parasitoid of B. oleae in wild olives (Copeland et al., 2004). In South Africa, the minor impact The African parasitoid Psyttalia lounsburyi (Silvestri) of B. oleae in wild olives is largely attributed to the action (Hymenoptera: Braconidae) was imported to California of the resident natural enemy fauna (Hancock, 1989), of as part of a biological control program directed at the olive which Utetes africanus (Sze´pligeti) is the dominant parasit- fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), a oid and P. lounsburyi is a significant component (Walton, recent arrival in the state (Collier and van Steenwyk, 2003; 2005). Despite its promise, little is known of the biology Rice et al., 2003). The literature suggests that P. lounsburyi of P. lounsburyi. It was identified nearly 100 years ago (Sil- may be an effective B. oleae parasitoid. In Kenya, it is the vestri, 1914), but little effort has been made to use it in the longstanding biological control programs for B. oleae in Europe (Greathead, 1976). These programs have focused * Corresponding author. Fax: +1 559 646 6593. almost exclusively on Psyttalia concolor (Sze´pligeti) since E-mail address: [email protected] (K.M. Daane). 1 Present address: Department of Horticulture, Oregon State University, the 1950s, when an efficient mass-rearing technique for this Corvallis, OR 97331-7304, USA. parasitoid was developed that uses the Mediterranean fruit

1049-9644/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.biocontrol.2007.08.010 80 K.M. Daane et al. / Biological Control 44 (2008) 79–89

fly, Ceratitis capitata (Wiedemann) (Tephritidae), in artifi- tephritid biological control agents, and a native species, cial diet (Clausen, 1978; Greathead, 1976). Rhagoletis fausta (Osten Sacken), which feeds on wild bit- Among the various parasitoids considered for B. oleae ter cherry fruit (Rosaceae). These non-target species repre- biological control in California (Sime et al., 2006a,b,c), sent the three major feeding niches of native California P. lounsburyi is especially attractive because there is tephritid larvae (flower heads of Asteraceae, galls, and evidence, again from the literature, that it is more of a fruits) (Foote et al., 1993). Given the challenges posed by specialist on B. oleae than other parasitoid species reared the large number of tephritid species in California, our from B. oleae. Its known geographic range—collections approach to assess the potential non-target host range of have been made from Kenya and South Africa—is entirely candidate parasitoids was to test a small number of tephri- contained within that of B. oleae (Nardi et al., 2005; Whar- tid species selected to span a broad range of phylogenetic ton and Gilstrap, 1983). Unlike other common braconid diversity and larval habitats, and to compare parasitoid parasitoids of B. oleae, such as P. concolor and Bracon celer behavioral responses and reproductive success relative to Sze´pligeti, which are often collected from Ceratitis species, the target species and its habitat. Second, we determined in field collections P. lounsburyi has been reared only from basic biological parameters of P. lounsburyi, including B. oleae (Narayanan and Chawla, 1962; Neuenschwander, adult longevity and fecundity, in order to compare its 1982; Wharton and Gilstrap, 1983; Wharton et al., 2000). potential with other braconids imported and evaluated The absence of other host records is not for lack of inves- for B. oleae biological control. The accumulated experience tigative effort. Likely alternative hosts have been reared gained during these studies will be helpful in developing throughout Africa during decades of foreign exploration field-release protocols and techniques for rearing P. louns- for natural enemies of various tephritid pests (Clausen, buryi on B. oleae. 1978; Clausen et al., 1965; Wharton, 1989; Copeland et al., 2004). That P. lounsburyi has not been reared from 2. Materials and methods C. capitata despite intensive efforts to find natural enemies of this pest (Wharton et al., 2000) and despite the fact that 2.1. Sources of insects and plants, and colony maintenance it can be cultured on C. capitata in the laboratory (Billah et al., 2005) suggests that other tephritid species are accept- Separate colonies of B. oleae and P. lounsburyi were able but not attacked. Many tephritid parasitoids use cues established at two University of California facilities, the from host plants to help locate their insect hosts (Messing College of Natural Resource’s Quarantine in Berkeley and Jang, 1992; Godfray, 1994). Therefore, the apparent and the Kearney Agricultural Center’s Insectary in Parlier, field specialization of P. lounsburyi on B. oleae might also California. Bactrocera oleae were reared on olive fruit include cues from the olive tree or fruit. using methods described by Sime et al. (2006b). In brief, Field evidence of specialization and efficacy alone is not adult flies were held in ventilated cages (50 cm3) that were considered sufficient for obtaining permission to release provisioned ad libitum with water and a 2:1 mixture of parasitoids exotic to the USA and often additional experi- honey and dry yeast extract (Fisher Biotech, Fairlawn, mental evidence of host specificity is also required (Hoel- NJ). Susceptible olives were exposed to the fly colony until mer and Kirk, 2005; van Driesche and Reardon, 2004). each fruit had 3–5 oviposition marks, typically <1 day, and Over 140 native tephritids inhabit California, including then removed to a separate rearing cage. The inoculated some endemic species (Foote et al. 1993). In California olives were held until the mature fly larvae exited the fruit there is also concern that introduced parasitoids of tephri- to pupate, upon which the puparia were collected and tid pests might attack beneficial tephritids used or consid- transferred to Petri dishes. The olives were collected from ered for biological control of weeds. Direct testing of orchards in Fresno County. Small to medium-size olives imported parasitoids’ responses to those tephritids is neces- (‘Mission’ or ‘Manzanillo’ cv.) were used for all experi- sary. Currently these include Chaetorellia succinea (Costa), ments, while olives of various cultivars, including ‘Ascolano’ which feeds in the flower heads of yellow starthistle, Cen- and ‘Sevillano,’ were also used for maintenance of the fly taurea soltitialis L. (Asteraceae) (Balciunas and Villegas, and parasitoid colonies. 2001), and Parafreutreta regalis (Munro), which forms The P. lounsburyi colony used in the Berkeley Quaran- stem galls in Cape ivy, Delairea odorata Lemaire (Astera- tine originated from adults that had emerged from B. oleae ceae) (Balciunas and Smith, 2006). These weeds can occur collected in wild (Olea europaea L. subsp. cuspidata (Wall. near olive trees, and this may promote encounters with par- ex G. Don)) and cultivated (ornamental) olives near Stel- asitoids of B. oleae. Yellow starthistle grows in low-eleva- lenbosch, South Africa, and were sent as puparia to the tion disturbed areas, and Cape ivy is widespread in Berkeley Quarantine in June 2004 and August 2005. After coastal habitats of California. a USDA-APHIS permit was granted to move P. lounsburyi We conducted a series of experiments to determine the out of Quarantine, experiments were conducted at the potential of P. lounsburyi as a biological control agent Kearney Agricultural Center using a P. lounsburyi colony for B. oleae in California and to proceed, if warranted, with established from 400 parasitoids shipped from the its field release. First, we evaluated the non-target impact USDA-ARS European Biological Control Laboratory in of P. lounsburyi on C. succinea and P. regalis, the two Montferrier, 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 the brought directly to the Berkeley Quarantine for Burguret Forest, Kenya, in 2005. The colony was initially experiments. maintained on B. oleae in olives, but starting in May 2005 the parasitoids were reared on C. capitata in artificial 2.2. Assessment of host range diet. These P. lounsburyi were reared on this medium for approximately 12 generations prior to arrival in California, Non-target tests were conducted in Quarantine using the at which time they were switched to rearing on B. oleae as South African P. lounsburyi. All experiments were con- described above. ducted in small cages (25 cm3) with glass fronts and screen Adult parasitoids of both P. lounsburyi colonies were sides. Each test consisted of two phases: a no-choice phase placed in ventilated cages (50 cm3) that were freely provi- in which parasitoids were offered only the non-target mate- sioned with fly-infested olives, water, and a honey–water rial, and a positive-control phase in which olives infested solution (50% by volume). Olives were infested with a mix- with B. oleae were added to the cages to confirm that ture of second and third instar B. oleae, as other Psyttalia P. lounsburyi were capable of oviposition into B. oleae. species oviposit into these stages (Mohamed et al., 2003; All plant materials were then incubated for at least 6 weeks Sime et al., 2006b). Inoculated olives were then transferred to allow adult parasitoid or fly emergence. If no adults to clean ventilated containers. Mature B. oleae larvae emerged, the plant material was dissected and fly puparia emerged from the fruit and dropped onto the bottom of were examined to determine if they had been parasitized. the container to pupate. These pupae were collected and Additional cages were set up as negative controls, in which transferred to Petri dishes. Unless stated otherwise, all non-target hosts and B. oleae were set up in parallel with experiments in Quarantine and at the Kearney Agricultural the no-choice tests and positive controls, and parasitoids Center took place in climate-controlled rooms (22 ± 2 C, were excluded from the treatment. 40% RH, 16:8 L:D supplemented by natural daylight) with The non-target fly species (C. succinea in the flower buds water and honey–water freely provided. of yellow starthistle, P. regalis in self-formed galls in Cape A P. regalis colony, used for non-target assessment, was ivy, and R. fausta in cherry fruit) were chosen in part to maintained at Berkeley Quarantine in summer and fall match B. oleae in size and because the galls, fruit, and buds 2004. The colony was established using stock (originally are comparable in size to olive fruit. Adequate host size from South Africa) maintained at the USDA—ARS quar- was considered important to avoid spurious parasitoid antine facility in Albany, California. Rearing methods used mortality due to insufficient host resources, while the size are described by Balciunas and Smith (2006). In brief, pot- of the infested structures was important to minimize nega- ted Cape ivy plants were placed in 32 · 45 · 96 cm sleeve tive results due to hosts that were buried beyond the reach cages with adult P. regalis for 4–7 days and then removed of parasitoid ovipositors. Most tephritid larvae had to racks under filtered daylight for gall maturation. Mature reached the third instar when the experiments were initi- galls were cut from the plants with a few cm of stem, and ated. All were presented in situ (i.e., in the gall, fruit, or placed in moist blocks of Oasis floral foam (Smithers- bud) in bouquets 10–20 cm in length, with the base in water Oasis, Cuyahoga Falls, OH) for adult fly emergence. and leaves removed to allow a clear view of wasp behav- A second non-target tephritid species, C. succinea, was iors. Buds of yellow starthistle were exposed to adult maintained using rearing methods based on those C. succinea 10–12 days before use in experiments. Dissec- described by Balciunas and Villegas (2001). Adult C. succi- tion of subsamples revealed that most flower heads were nea were reared from yellow starthistle flowers collected in infested, and that over 60% of the larvae were in the third Contra Costa and Yolo counties from July through Octo- instar. Cape ivy galls were harvested 20–27 days after expo- ber 2004. The emerging adult flies were caged and supplied sure to P. regalis. Subsamples of bitter cherries were dis- with water and a mixture of dry yeast extract and honey. sected to ensure that the majority of R. fausta reached To produce flower heads infested with groups of same-aged the third instar. larvae, potted plants were pruned of all but the suitable In the first (no-choice) phase of the experiment (first young flower buds, up to a week before expected anthesis, 48 h), a bouquet of the non-target plant material was and then placed in a cage with the reared adult flies (6- to placed in each cage. The amount of parasitoid and host 14-day-old) for 2 days. The plants were then moved to a material in each replicate depended on availability of mate- greenhouse (23 ± 3 C) to rear the larvae, which were used rial and consisted of 6–15 mated female P. lounsburyi, in non-target studies when they reached the appropriate which were previously exposed to B. oleae, with either stage. 8–10 galls (P. regalis), 10–20 flower heads (C. succinea), The third non-target species, the native black cherry or 20 fruit (R. fausta) in each cage. Each replicate began fruit fly, R. fausta, is a univoltine species found in ripening between 8:00 and 9:30 AM. The second (positive-control) bitter cherry, Prunus emarginata (Douglas ex Hooker) phase began at 9:00 AM on the third day, when 8–10 olives Eaton, in late summer and fall. Branches bearing fruit with (‘Manzanillo’, ‘Mission’, or ‘Sevillano’ cultivars) infested evidence of R. fausta infestation (oviposition scars) were with 5–10 third-instar B. oleae were placed in the cage at collected on the western slope of the Sierra Nevada moun- a height level with the non-target material. This phase tains (Fresno County) in August and September 2004 and lasted 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- At the start, four female P. lounsburyi were placed in a control phases, the parasitoids were observed for 10 min at ventilated cage (30 cm3) along with a fresh-cut olive branch 10:00, 13:00, and 16:00 h. The number of P. lounsburyi in (in water) for a 1 day acclimation period. Thereafter, four contact with the galls, flower heads, or fruit was recorded olives of each age category (20 total) were added for a 2 under three categories: investigating, probing, or incidental day oviposition period. Olives containing B. oleae hosts contact. ‘‘Probing’’ was defined as the insertion or of the same age were grouped in a small Petri dish (5-cm attempted insertion of the ovipositor. ‘‘Investigating’’ was diameter), and the five small Petri dishes were then placed defined as a walk with slightly lowered antennae, inter- inside a large Petri dish (15 cm diameter). To quantify the rupted by frequent stops with the antennae raised. Other parasitoid’s searching activity on different host stages, two behaviors on the host material, such as standing motion- 30 min observation sessions were made, one in the morning less, grooming, or brief contact, were recorded as incidental and the other in the afternoon on the first day of exposure. contact. During these sessions, the number of parasitoids observed At the end of the positive-control phase, fruit flies were on olives was recorded at 3 min intervals to estimate the separated and incubated for a minimum of 6 weeks. Bou- relative amount of parasitoid searching time on different quets of galls, still maintained on stems in water, were con- host stages. After the exposure period the olives were held fined in vials with mesh windows, while flowers and fruit at 25 ± 2 C to rear either adult parasitoids or flies. were removed from stems and held in plastic vials (flowers) or paper cups (fruit) with mesh lids. The numbers of adult 2.4. Pre-imaginal developmental rates at different constant flies and parasitoids that emerged were recorded. Galls and temperatures flower heads were later dissected and the number of dead larvae, pupae, and adults inside recorded. Fruit were dis- Developmental rates (egg to adult) of P. lounsburyi were sected only in replicates lacking parasitoid emergence. assessed at three constant temperatures (17.9 ± 0.7, The negative controls were treated in a similar manner. 23.9 ± 1.1, and 27.1 ± 1.2 C) in temperature cabinets. Unemerged puparia were dissected after the 6 week incuba- Olives infested with second and third instar B. oleae were tion period to determine if they contained unparasitized exposed to ovipositing P. lounsburyi for 24 h. An over- host pupae or encapsulated eggs or cadavers of parasitoids. abundance of B. oleae was presented (about 10 infested Dead fly pupae with recognizable form were assumed to be olives per female wasp) to limit the level of superparasitism. unparasitized because the Psyttalia species we studied After exposure to P. lounsburyi, the infested olives were killed the host directly after formation of the puparium. apportioned evenly among temperature treatments. A Petri Unencapsulated parasitoid eggs and first instars could dish of water was placed inside each temperature cabinet to not be detected with this technique. Therefore, host maintain the humidity. The containers were checked twice remains that had clearly not differentiated into pupae and daily (morning and afternoon) to record the number and also lacked later parasitoid instars were treated as ‘‘unde- sex of emerging parasitoids. Twenty replicates were made termined’’ and excluded from the analysis. for each temperature.

2.3. Host stages preferred for oviposition 2.5. Adult longevity

The preference of P. lounsburyi for olives infested with Adult male and female longevity were measured at different B. oleae stages was examined. The experiment seven temperatures (15.2 ± 0.3, 21.9 ± 0.2, 24.8 ± 0.4, was conducted at the Kearney Agricultural Center (using 27.8 ± 0.2, 30.1 ± 0.2, 32.0 ± 0.5, and 34 ± 0.5 C). Newly Kenyan P. lounsburyi). To produce an age series of B. emerged parasitoids were isolated in glass vials (5-cm oleae, fresh olives were exposed to flies for 2 h every 2–3 long · 1-cm diameter, with a mesh lid) and randomly days and then held at 25 ± 1 C. Olives used had 3–5 ovi- assigned to a temperature treatment. The parasitoids were position marks. Immature stages inside the olives were checked daily for mortality. At each temperature, 10 male presented to the parasitoids at 1, 3, 6, 8, and 10 days, and 10 female parasitoids were tested. using a 2 days exposure period each time, to create five Adult female longevity was also compared among five host age categories of 1–2, 3–4, 6–7, 8–9, and 10–11 days. treatments of different host and/or food provisions: (1) A sub-sample of olives from each set was dissected before olives containing hosts, honey–water (50% by volume), and after each test to determine which host stages were and water; (2) uninfested olives, honey–water and water; present. Under these conditions, 1–2 days post exposure (3) honey–water and water only; (4) water only; and (5) olives contained mostly eggs and a few first instars; 3–4 no provisions. Newly emerged females were collected daily, days post exposure olives contained first and second transferred to a small container with males, supplied with instars; 6–7 days post exposure olives contained second water and honey–water, held for 2 days to mate, individu- instars and young third instars; 8–9 days post exposure ally isolated in ventilated plastic containers (15 cm diame- olives contained mostly third instars; and 10–11 days post ter · 6 cm deep), and then randomly assigned to a exposure olives contained mature third instars and occa- treatment. Olives (four per container) were replaced every sionally puparia. other day. Where olives with hosts were offered, each olive K.M. Daane et al. / Biological Control 44 (2008) 79–89 83 had 5–10 fly oviposition marks (20–40 larvae for each 2 day target host material was observed at low rates in all tests interval) and the fly larvae were at a suitable stage (second but was not different between the no-choice and choice and third instars) for parasitoid oviposition. Parasitoids phases of the studies (Fig. 1A). Neither probing (Fig. 1B) were checked daily for mortality. Ten replicates were made nor investigation behaviors (Fig. 1C) were observed in per sex per treatment. Experiments were conducted at the the no-choice phase of the experiment, except for two inci- Berkeley Quarantine using South African P. lounsburyi. dences of investigation behavior observed in the choice phase on Cape ivy galls. The parasitoids did, however, 2.6. Fecundity investigate and probe olives with B. oleae larvae in the choice phase of the experiment (Fig. 1), and offspring were Lifetime fecundity was investigated at the Kearney Agri- recovered from these hosts (Table 1). Psyttalia lounsburyi cultural Center, using the Kenyan P. lounsburyi. Newly investigated olives more often than yellow starthistle emerged female and two male P. lounsburyi were placed (Fig. 1B, one-way ANOVA, F = 7.64, df = 2, 32, in a ventilated cage (15 · 15 · 20 cm3). Ten infested olives, P = 0.002; Tukey’s HSD, P < 0.05) and bitter cherry fruit each containing 3–5 second or third instar B. oleae, were (Fig. 1B, one-way ANOVA, F = 10.00, df = 2, 14, continuously provided during the adult parasitoids’ lifetime. The olives were presented on a Petri dish (8.5 cm diam), raised 5 cm above the cage floor. Every 2 days, the exposed olives were replaced with new olives. Male P. lounsburyi were removed after 4 days. The exposed olives were isolated according to exposure dates, and incubated as described previously. Emerging flies or parasitoids were recorded daily. Four replicates were made of each treatment, with 4–9 females in each replicate. A sub-sample of newly emerged female P. lounsburyi from the colony was dissected to estimate egg load. All tested female parasitoids were immediately dissected after death to determine the number 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 time were analyzed using one-way ANOVA, and treatment means 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 longevity data were analyzed with the Cox survival analysis model and Kaplan-Meier estimator (Systat 12, Systat Soft- ware, Inc., San Jose, CA). Female and male longevity were compared using the parametric model function with gender as the strata and temperature as the covariate; the output provided the Mantel–Haenszel log-rank test for strata sep- aration (Chi-square) and a Z-test for impact of the covariate. Temperature and food provision effects on adult female longevity were similarly compared for all possible pair-wise treatment comparisons, using the nonparametric model function. In these analyses, the experiment-wide Fig. 1. Comparison of mean (±SEM) number of responses of female P. error rate used was alpha’ = alpha/n, where alpha = 0.05 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-m and n is the number of possible pair-wise comparisons. observation periods during a subsequent 48-h choice phase (NT and B. oleae). The responses were categorized as: (A) incidental contact without 3. Results any apparent response to hosts; (B) investigation behavior indicating awareness of host presence; and (C) probing the substrate with the 3.1. Assessment of host range ovipositor. The non-target hosts were Chaetorellia succinea in yellow starthistle flower heads, Parafreutreta regalis in Cape ivy stem galls, and Rhagoletis fausta in bitter cherry fruit. Bactrocera oleae were offered in Psyttalia lounsburyi showed almost no interest in the olives. Different letters above each group of bars indicate significant non-target hosts (Fig. 1). Incidental contact with the non- differences (one-way ANOVA, followed by Tukey’s HSD, P < 0.05). 84 K.M. Daane et al. / Biological Control 44 (2008) 79–89

Table 1 Number of parasitized and unparasitized offspring reared from B. oleae and non-target hosts during the P. lounsburyi host-specificity study Non-target Offspring reared from hosts exposed to parasitoids (no-choice and choice Offspring reared without exposure to parasitoids (negative host phases) control) 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 11 509 P. regalis 10 172 0 746 39 5 60 7 664 R. 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.

P = 0.003; Tukey’s HSD, P < 0.05) in both phases of the study. When P. regalis in Cape ivy galls was offered without choice, the parasitoids investigated the galls less than the 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 the choice phase the difference in P. lounsburyi’s responses to galls or olives was not significant. Throughout the non-target host study, probing was observed only in olives but was rare even in the target host and was higher only when compared 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 the olives differed across age categories tested (F = 5.35, df = 4, 95, P < 0.001) (Fig. 2A). More parasitoids were observed 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 were reared per replicate. Parasitoids were reared from all host age categories, but more were reared from the 3- to 9-day-old larvae (late 1st instar to 3rd instar) than from 1- 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 Fig. 2. Host-stage preference as (A) mean percentage (±SEM) adult Developmental time (egg to adult) of P. lounsburyi was female Psyttalia lounsburyi on olives containing hosts of a given age category during timed observation intervals (linear regression: y = 4.05x– significantly affected by temperature and sex (temperature: 6.74, r2 = 0.818; F = 13.56, df = 1,4, P = 0.034) and (B) as mean percent- F = 2081.6, df =2, P < 0.001; sex: F = 14.2, df =1, age (±SEM) of P. lounsburyi offspring that emerged from different host P < 0.001; temperature · sex: F = 2.6, df =2, P = 0.07). age categories (log normal ‘‘peak’’ regression: y = a exp(À0.5(ln(x/x0)/ 2 Under each temperature the developmental time of female b) ), where a, b, and x0 are 35.76, 0.51, and 5.34, respectively, F = 37.66, P. lounsburyi was significantly longer than that of males. df = 2,4, P = 0.026). Within in graph, different letters near each mean indicate significant differences (one-way ANOVA, P < 0.05). Developmental rates for both male and female wasps increased with temperature (Fig. 3). P < 0.001) and for that reason female and male data were 3.4. Adult longevity analyzed separately. Adult longevity ranged from an average 46.8 ± 3.4 days (15 C) to <1 day (34 C), generally Across all temperature treatments, adult females lived decreasing with increased temperature for both male and longer than males (Fig. 4A). Temperature treatments female P. lounsburyi although at 25 C both longevities had a significant impact (covariate analysis: Z = 8.669, were lower than expected (Table 2). Survival analysis is K.M. Daane et al. / Biological Control 44 (2008) 79–89 85

Table 2 Longevity (±SEM) of adult female and male P. lounsburyi when held at constant temperatures and provisioned with honey and water Temperature (C) Adult longevity (in days) Female Male 15 46.8 ± 3.4 25.7 ± 4.7 22 22.4 ± 3.8 11.8 ± 2.2 25 10.7 ± 1.7 5.3 ± 1.0 28 14.3 ± 1.9 11.2 ± 1.2 30 11.2 ± 1.7 9.2 ± 0.7 32 2.4 ± 0.2 2.0 ± 0.2 34 <1.0 <1.0

presented for adult females only, although males showed a similar pattern. At 34 C, no adults survived to the first observation period (day 1) and these data were not Fig. 3. Mean developmental time (egg to adult) (±SEM) of Psyttalia included in the analysis. Adult females lived longer at lounsburyi at different constant temperatures. Different letters over each 15 C and progressively shorter with increasing tempera- bar within the same sex (female indicated by capital and male by lower tures (Fig. 4B). Longevity was affected by provisioning case letters) indicate significant differences (one-way ANOVA, P < 0.05). treatment, as indicated by survival analysis (Fig. 5). Female P. 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 infested with 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. The ten olives used per 2 day exposure period contained a total of 27.1 ± 1.5 (n = 56) host larvae based on dissection of random samplings of infested olives; therefore most of the presented hosts were unparasitized. Dissection of

Fig. 4. Survivorship curves for adult Psyttalia lounsburyi for (A) male and females with data combined across all temperatures showing females are longer 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 letters Fig. 5. Adult female Psyttalia lounsburyi longevity given different food following each strada in the keys indicate a significant difference in provisions. Different letters following each provision treatment in the key survival analysis pairwise comparisons for gender (alpha = 0.05) and indicate a significant difference in survival analysis pairwise comparisons temperature (alpha = 0.0033). (alpha = 0.005). 86 K.M. Daane et al. / Biological Control 44 (2008) 79–89

investigated and probed bitter cherries at high rates (unpublished data). Further evidence for strong olive habitat specificity is provided by probing and reproduction of P. lounsburyi on C. capitata larvae inserted into olive fruit (R. Wharton, personal communication). Taken with evidence from African field surveys, these data argue that P. lounsburyi specializes on B. oleae by vir- tue of either a limited range of acceptable host species or searching behavior that is limited to olives. Host specificity arises from behavioral and physiological adaptations that limit the range of organisms that parasitoids will attack and utilize as hosts, and behavioral adaptation precedes physiological adaptation in the evolution of host specificity (Futuyma and Moreno, 1988). Host-habitat specificity may be more advantageous in a biological control agent than Fig. 6. Mean number (±SEM) of Psyttalia lounsburyi offspring produced physiological specificity, as it may serve to focus host per parasitoid over her lifetime when provided with unlimited quantifies of searching on olives, thereby improving parasitoid efficiency B. oleae larvae. in addition to reducing potential risk to non-target hosts. For that reason, information developed from laboratory exposed fruit after all flies and parasitoids had emerged cage studies may not reflect the actual host range. Also, revealed that percentage of dead puparia was low only three non-target tephritids were evaluated. We had (2.48 ± 0.47%, n = 27). Thus, the results appear to reflect hoped to test more native tephritid species; however, two the maximum reproductive potential of the parasitoid years of field collections for known California native spe- under the given laboratory conditions, rather than parasit- cies recovered insufficient material that coincided with par- ized but undeveloped P. lounsburyi. asitoid availability, and we were unable to develop colonies Female P. lounsburyi in this experiment survived of those that were recovered. Moreover, P. lounsburyi 41.6 ± 3.5 days (range 12–70 days). Newly emerged females response to B. oleae was far lower than that recorded for (<12 h) contained few mature eggs (1.17 ± 0.56 per female, other braconid species tested in Berkeley Quarantine (Sime n = 12). Egg maturation appeared to proceed quickly. et al., 2006b,c), suggesting that experimental conditions Females dissected 24–48 h after emergence and deprived were not ideal for normal behavior. Several authors have of hosts contained 17.2 ± 2.1 mature eggs (n = 13). The emphasized the importance of in situ parasitoid behaviors parasitoids began to oviposit on the second day after emer- and host microhabitat in non-target evaluations (Messing, gence and reached peak oviposition tempo at 6–10 days 2001). For example, Duan and Messing (1997) suggest that (Fig. 6). Oviposition activity decreased thereafter and the spheroid shape of a non-target host substrate, such as nearly stopped after 30 days, although females still con- galls, may be similar enough to small fruit to elicit host- tained 16.6 ± 3.1 eggs at death. Of the 20 females tested, searching behaviour in opiine fruit fly parasitoids while three did not produce any offspring. flowerheads of Asteraceae shrubs housing flowerhead tephritids have no resemblance to the host fruits of frugivorous tephritids. 4. Discussion Studies of P. lounsburyi biological parameters provide some important information for field release and insectary Results of non-target assessment provide evidence that production guidelines. Although P. lounsburyi has been P. lounsburyi has a narrow host range or behaviors that successfully reared from B. oleae attacked in the early third limit host searching to olives or similar fruit. The experi- instar (Billah et al., 2005), its preference for different host mental method used was relatively conservative, as the stages and relative success in parasitizing them have not small cage size promoted high encounter rates with the been investigated, nor has its behavior been described when infested plant material regardless of the type of long-range offered B. oleae feeding in fruit. Female P. lounsburyi show orientation cues used by the parasitoid to locate a particu- a preference for older third-instar host larvae, compared lar host-plant complex. The lack of investigation or prob- with egg to second instar stages (Fig. 2A). However, there ing in bitter cherries, the most likely alternate habitat was a poor correlation between the host stages attacked tested to induce a searching response from a parasitoid and host stages producing the most offspring (Fig. 2B). adapted to a frugivorous host, provides support for a This discrepancy may be explained as the outcome of two reduced risk to non-target tephritids by P. lounsburyi.In independent factors: an increased ability to locate older lar- concurrent tests under the same conditions, other braconid vae in the fruit, but a decreased ability to oviposit in older olive fly parasitoids, P. concolor and Bracon celer Sze´pligeti, larvae in cultivated olives. Many parasitoids locate hidden and braconids that attack other tephritids, Diachasmimor- hosts through substrate vibrations (Glas and Vet, 1983; pha kraussii (Fullaway), and D. longicaudata (Ashmead), 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, locates ment. These results do not bode well for P. lounsburyi hosts through vibrotaxis (Canale and Loni, 2006). The establishment in the inland valleys. However, because these suites of searching behaviors exhibited by P. concolor and data were collected using hosts feeding on picked fruit, they P. lounsburyi on infested olives are the same (H. Nadel, may not reflect performance in the field. Collected olives personal observation). Psyttalia concolor responds more tend to dry out and shrivel when held at temperatures strongly to the third instar than the second instar of C. cap- above 25 C, which may impede parasitoid development itata in experimental arenas (without fruit); presumably (Sime et al., 2007). Fruit still on a tree would remain turgid because the larger instar produces stronger or more fre- at even higher temperatures. Therefore, the temperature quent vibrations while feeding (Canale and Loni, 2006). development rates are better used to compare P. lounsburyi Psyttalia concolor does not reject either of these instars to other parasitoid species tested against B. oleae using the once it locates them, and also develops with equal success same experimental conditions. The developmental rates in both (Canale and Loni, 2006). For P. lounsburyi, the and mortality of P. lounsburyi are similar to those observed third instar induces more searching behavior, but the third for other B. oleae parasitoids tested, including D. kraussii instar B. oleae feed deeper than the second instar in culti- and D. longicaudata (Sime et al., 2006c), P. ponerophaga vated olives, which are much fleshier than wild olives. (Sime et al., 2007), and P. concolor (Sime et al., 2006b). For most of the third instar, therefore, the larvae are This finding indicates that P. lounsburyi would perform beyond the reach of the short P. lounsburyi ovipositor as favorably as other species being considered for use in (<2 mm) (H. Nadel, unpublished data). California, based on temperature tolerances, across the The results of our P. lounsburyi host-age preference range of climates found in the olive-growing regions. study show a clear discrepancy between the age categories Similarly, overall patterns of adult longevity are compa- on which adult parasitoids most commonly searched and rable to these other species, apart from P. concolor, which probed (Fig. 2A) and from which parasitoids were reared can live for long periods relative to the other species at tem- (Fig. 2B). These data lend further support to our hypothe- peratures of 30 C or higher (Sime et al., 2006b). With sis that the shorter ovipositor of P. lounsburyi cannot reach respect to provisioning, the decrease in adult longevity the larger host larvae that feed deeper inside the cultivated when host-infested fruit were available suggests that the olive—although these host stages may be acceptable and parasitoids expended energy in attempting or succeeding even preferred. It is also possible that the older larvae are to parasitize hosts. This suggests that the parasitoids were parasitized but better able to encapsulate the parasitoid able to distinguish host-infested olives from those lacking egg. These results differ from those found for other Psyttal- hosts. Identical patterns were observed in P. concolor (Sime ia species that were tested similarly using an age series of B. et al., 2006b), D. kraussii, and D. longicaudata (Sime et al., oleae larvae. Psyttalia ponerophaga (Silvestri) showed no 2006c), though not in P. ponerophaga, which responded discrepancy between preference and reproductive success, equally to all three treatments in which honey was provided but rather an increase in both with larval age (Sime (Sime et al., 2007). The argument could also be made that, et al., 2007). A discrepancy was observed in P. concolor without hosts, these Psyttalia species resorb eggs to utilize (Sime et al., 2006b), but it is different than that found in reserves (Greathead 1976). P. lounsburyi, and also differs from the predictions of In contrast, P. lounsburyi fecundity was lower than other Canale and Loni (2006).InSime et al. (2006b), P. concolor species tested as B. oleae parasitoids under similar experi- preferred to oviposit into second and young third instars, mental conditions, including P. ponerophaga (18.7 ± 2.9 but most larvae were reared from hosts attacked as third offspring per female) (Sime et al., 2007), two laboratory instars. Here, the discrepancy was proposed to result from strains of P. concolor (22.5 ± 5.1 and 28.7 ± 4.1 offspring the relatively short ovipositor of Psyttalia species and the per female) (Sime et al., 2006b), and two Diachasmimorpha peculiar pre-pupation behavior of B. oleae (Sime et al., species (23.6 ± 5.3 and 22.7 ± 5.5 offspring per female) 2006b, 2007). In the pulp of cultivated olives, which is typ- (Sime et al., 2006c). This result is somewhat surprising ically much deeper than the length of a Psyttalia oviposi- because we expected P. lounsburyi, as an adapted specialist, tor, third-instar B. oleae generally feed closest to the pit, to perform better on B. oleae feeding in fruit compared but shortly before pupation the older third instar tunnels with the more polyphagous P. concolor or the two Diacha- near the surface to create an exit ‘‘window’’. The late third smimorpha species for which B. oleae is an entirely novel instar may, therefore, be the most vulnerable to attack by host. One hypothesis to explain low parasitism rates again some Psyttalia species. concerns the short ovipositor of P. lounsburyi relative to Temperature tolerances will also be an important factor the feeding depth of hosts within cultivated olives. Wild in the establishment and performance of parasitoids in Cal- fruit are small, around 7–8 mm in diameter, while culti- ifornia because B. oleae infestations are found in both the vated varieties are typically 2–3 cm in diameter (Bartolini relatively cool coastal and hot inland areas. In our investi- and Petruccelli, 2002; Tzanakakis, 2003). In the field, it gation of pre-imaginal developmental rates, a relatively has been reported that P. lounsburyi is not found as fre- high mortality of parasitized hosts (70–80%) was observed quently in cultivated olives as in wild olives. A survey in at the highest temperature (27 C), which suggests that P. South Africa produced 22 individuals from wild olives lounsburyi has a relatively low upper threshold for develop- and 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 has References a longer ovipositor (3 mm, instead of <2 mm) and favors mature larvae, was about equally abundant in wild and cul- Balciunas, J.K., Smith, L., 2006. Prerelease efficacy assessment, in tivated olives. The hypothesis is also supported by the quarantine, of a tephritid gall fly being considered as a biological control agent for Cape ivy (Delairea odorata). Biological Control 39, results of the longevity experiment comparing responses 516–524. to different provisioning regimes. Balciunas, J.K., Villegas, B., 2001. Unintentionally released Chaetorellia The poor reproductive performance of P. lounsburyi succinea (Diptera: Tephritidae): Is this natural enemy of yellow compared with the other Psyttalia species indicates that starthistle a threat to safflower growers? Environmental Entomology there are confounding factors in addition to the shortness 30, 953–963. Bartolini, G., Petruccelli, R., 2002. Classification, Origin, Diffusion and of its ovipositor. One alternative explanation for our History of the Olive. Food and Agriculture Organization of the United results compared with the field observations hinges on Nations, Rome. the chemical cues used to orient to and identify host larvae Billah, M.K., Kimani-Njogu, S.W., Overholt, W.A., Wharton, R.A., and the host/plant complex. Domestic olives differ chemi- Wilson, D.D., Cobblah, M.A., 2005. The effect of host larvae on three cally from wild olives (Massei and Hartley, 2000). These Psyttalia species (Hymenoptera: Braconidae), parasitoids of fruit- infesting flies (Diptera: Tephritidae). International Journal of Tropical differences could disrupt the parasitoid’s host-searching, Insect Science 25, 168–175. host-identification, or ovipositional behaviors, or impede Canale, A., Loni, A., 2006. Host location and acceptance in P. concolor: larval development. Other differences in chemistry are role of host instar. Bulletin of Insectology 59, 7–10. likely between picked olives, as used in these experiments, Clausen, C.P., 1978. Introduced Parasites and Predators of Arthropod and fruit still on the tree. The more specialized P. louns- Pests and Weeds: A World Review. United States Department of Agriculture, Agriculture Handbook No. 480, Washington, D.C. buryi is expected to be especially sensitive to changes in Clausen, C.P., Clancy, C.W., Chock, Q.C., 1965. Biological control of plant chemistry, and to have innate, relatively inflexible the oriental fruit fly (Dacus dorsalis Hendel) and other fruit flies in responses to semiochemicals used to find hosts and plants Hawaii. U.S. Department of Agriculture Technical Bulletin 1322, (Vet and Dicke, 1992). 1–102. Previously, no effective rearing method had been devel- Collier, T.R., van Steenwyk, R.A., 2003. Prospects for integrated control of olive fruit fly are promising in California. 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