BIOLOGICAL CONTROL-PARASITOIDS AND PREDATORS

Overwintering Survival of Olive Fruit Fly (Diptera: Tephritidae)and Two Introduced Parasitoids in California

XIN-GENG WANG,1 KARMIT LEVY,1 HANNAH NADEL,2 MARSHALL W. JOHNSON,3

ARNAUD BLANCHET,4 YAEL ARGOV,5 CHARLES H. PICKETT,6 AND KENT M. DAANE1,7

Environ. Entomol. 42(3): 467Ð476 (2013); DOI: http://dx.doi.org/10.1603/EN12299

ABSTRACT The overwintering survival and development of olive fruit ßy,Bactrocera oleae (Rossi),and the endoparasitoids, Psyttalia humilis Silvestri and P. lounsburyi (Silvestri), were investigated atsites in CaliforniaÕs interior valley and coastal region. In the interior valley, adult ßies survived up to4Ð6 mo during the winter when food was provided. Adult female ßies could oviposit in late fall andearly winter on nonharvested fruit and, although egg survival was low (0.23Ð8.50%), a portion of theoverwintered cohort developed into adults the following spring; percentage of survival was negativelycorrelated to daily minimum temperature.P. humilis andP. lounsburyi successfully oviposited into hostlarvae in late fall, and their progeny developed into adults the following spring, although with a lowpercentage (0Ð11.9%) survivorship. Overwintering survival of puparia of the olive fruit ßy andimmature larvae of P. humilis and P. lounsburyi (inside host puparia), buried in the soil, were testedat an interior valley and coastal site. Survival of olive fruit ßy ranged from 0 to 60% and was affectedby the trial date and soil moisture. Overwintering survival of both the fruit ßy and tested parasitoidswas lower at the colder interior valley than the coastal site; P. humilis immature stages had the highestmortality levels while B. oleae pupae had the lowest mortality levels. The spring emergence patternof the tested insectswaswellpredictedbyadegree-daymodel.Wediscuss factorspotentially impedingestablishment of introduced olive fruit ßy parasitoids in California and elsewhere.

KEY WORDS Bactrocera oleae, biological control, parasitoid, Psyttalia, overwintering

Successful establishment of arthropod natural ene-mies requires synchrony of the imported natural en-emy with the targeted pestÕs seasonal biology andhabitat (DeBach and Bartlett 1964). An analysis ofregional and national reviews of biological controlprograms suggests improper climatic match ac-counted for 34.5% of failures (Stiling 1993). Poor over-wintering survival of an introduced natural enemy isone of the major limitations imposed by climate onnatural enemy establishment and success (Boivin et al.2006, Jenner et al. 2010). Therefore, understanding theoverwintering survival of introduced natural enemiesis central to assessing their potential to establish innew geographic ranges. Here, we investigated theoverwintering survival of natural enemies imported to

California for control of the olive fruit ßy, Bactroceraoleae (Rossi). Before our studies, classical biologicalcontrol ofB. oleae had been investigated for over 80 yrin the Mediterranean basin. A braconid larval parasi-toid, Psyttalia concolor (Szepligeti) was introducedfrom North Africa and released widely, but with lim-ited success (Raspi and Loni 1994, Miranda et al.2008). Although detailed studies are lacking on itsoverwintering biology, Loni (1997) speculated thatthe failure of P. concolor to establish in some regionsof the Mediterranean was due, in part, to winter cli-mate extremes.B. oleae is a major olive pest worldwide and has been

the target of numerous biological control programs(Tzanakakis 2006, Daane and Johnson 2010, Argov etal. 2012). Believed to be native to sub-Saharan Africa,the olive fruit ßyÕs range expanded naturally, probablyafter the domestication of olives, into the Mediterra-nean basin, South and Central Africa, SouthÐCentralAsia, and more recently into California and north-western Mexico (Zygouridis et al. 2009, Nardi et al.2010). A renewed interest in classical biological con-trol occurred with the discovery of B. oleae in Cali-fornia (Daane et al. 2011). Numerous braconid para-sitoids were screened for their potential as B. oleaenatural enemies, including P. concolor (Sime et al.2006b), Psyttalia lounsburyi (Silvestri) (Daane et al.2008), Psyttalia humilis Silvestri (Wang et al. 2011b),

1 Department of Environmental Science, Policy and Management,137 Mulford Hall, University of California, Berkeley, CA 94720-3114.

2 U.S. Department of AgricultureÐAnimal and Plant Health Inspec-tion Service, Plant Protection and Quarantine, 1398 W. Truck Rd.,Buzzards Bay, MA 02542.

3 Department of Entomology, 900 University Ave., University ofCalifornia, Riverside, CA 92521.

4 USDAÐARS, European Biological Control Laboratory, 810, Ave-nue du Campus Agropolis, 34980 Montferrier, France.

5 Plants Production and Marketing BoardÐCitrus Division, IsraelCohen Institute for Biological Control, P.O. Box 54, Bet Dagan, Israel50250.

6 California Department of Food and Agriculture, 3288 Mead-owview Rd., Sacramento, CA 95832.

7 Corresponding author, e-mail: kdaane@ucanr.edu.

0046-225X/13/0467Ð0476$04.00/0 � 2013 Entomological Society of America

Bracon celer Szepligeti (Sime et al. 2006a, Nadel et al.2009), Psyttalia ponerophaga (Silvestri) (Sime et al.2007), Fopius arisanus (Sonan) (Sime et al. 2008),Diachasmimorpha kraussii Viereck, and Diachasmi-morpha longicaudata (Ashmead) (Sime et al. 2006c).At present, two African larval endoparasitoids, P.lounsburyi and P. humilis, have been released in Cal-ifornia. All these larval endoparasitoids emerge asadults from host puparia. To facilitate insectary pro-duction, both parasitoid species were reared on theMediterranean fruit ßy (Medßy), Ceratitis capitata(Wiedemann), with large statewide release of P. hu-milis (Yokoyama et al. 2010, 2011), whereas fewer P.lounsburyiwere released and at coastal California sitesonly (X.G.W., unpublished data). Both parasitoid spe-cies have successfully overwintered at coastal releasesites (K.M.D., unpublished data), although data fromthe release programs has not yet shown either para-sitoid to successfully overwinter in CaliforniaÕs inte-rior valley, where summer temperatures are higherand winter temperatures are colder than at coastalolive growing regions.

In contrast, there is ample evidence that B. oleaemayoverwinteras a larvaoradult inCaliforniaÕs south-ern and coastal regions, where there are mild wintertemperatures.B. oleae larvae have been collected fromoverwintered fruit in the spring and baited trapscaught adult ßies in both winter and early springperiods (Yokoyama et al. 2006, Burrack et al. 2011). InCaliforniaÕs interior valley, baited traps also caughtlow numbers of adult B. oleae in winter and earlyspring (Yokoyama et al. 2006), but it is unknown howlong the adult survives in the Þeld. The pupa is themost likely overwintering stage. In late fall, maturelarvae commonly exit fruit to pupate in the soil(Tzanakakis 2006), and typically the ßy will overwin-ter outside of the fruit and just under the soil surfaceinside a puparium. Nevertheless, it is unknown if B.oleae can overwinter in CaliforniaÕs interior valley asa late-instar or pupa in the soil. The goals of this study,therefore, were to compare the overwintering survivalof B. oleae, P. humilis, and P. lounsburyi in California.SpeciÞcally, in interior valley sites we investigatedadultB. oleaeoverwintering survival in Þeld cages, andsurvival of ßy eggs or larvae in fruit. Concurrently, wetested the survival of both parasitoid species as im-matures (inside a host) on olive fruit. In a coastal siteand two interior valley sites, we investigated the over-wintering survival of B. oleae puparia when lightlyburied in the soil, and both parasitoid species (insidea host). The research is discussed with respect to thesurvival patterns among these species and those fac-tors that may impede establishment of the introducedparasitoids.

Materials and Methods

Insect Cultures. Laboratory colonies of B. oleaewere maintained on olives in insectaries (24 � 2�C, aphotoperiod of 16:8 [L:D] h, 40Ð60% relative humid-ity ]RH]) at the University of California (UC), Kear-ney Agricultural Research and Extension Center

(Kearney) in Parlier, CA, and the UC Oxford TractFacility in Berkeley, CA (UC Berkeley). Olives werecollected from an insecticide-free orchard at Kearney.Rearing procedures were described previously forthe ßy (Wang et al. 2009a), and P. humilis and P.lounsburyi (Wang et al. 2011b). The B. oleae colonywas established in 2003 with adults reared from olivescollected in Davis, CA, and Þeld-collected ßies wereadded to the colony each year to maintain colonyvigor. Adult ßies were held in Bug Dorm2 cages (Bio-Quip, Rancho Dominguez, CA) provisioned with wa-ter, honey, and hydrolyzed yeast (Fisher Biotech,Fairlawn, NJ). Olives were exposed to gravid femaleB.oleae until each fruit had 3Ð5 oviposition stings. In-fested olives were then distributed over a piece of wiremesh that rested 2 cm above a rectangular (36 by 18by 10 cm) plastic tray. Larvae matured in 9Ð12 d,exited the fruit and dropped onto the tray, wherepuparia were collected and then placed into a cleanholding cage for emergence of adult ßies.

Parasitoid colonies were maintained at the Kearneyinsectary on B. oleae infested olives. The P. lounsburyicolonywasestablishedwithadults reared fromB.oleaecollected on wild olives in 2002 in the Burguret Forest,Kenya (Daane et al. 2008); with new stock from thissame region added to the colony yearly from 2002 to2005. TheP. humiliscolony was established with adultsreared fromB. oleae collected on wild olives in 2008 inNamibia. This parasitoid was previously referred to asP. concolor (Rehmen et al. 2009) and P. cf. concolor(Yokoyama et al. 2008). Both parasitoid colonies weremaintained on Medßy larvae in artiÞcial diet at theU.S. Department of AgricultureÐAgriculture ResearchServices (USDAÐARS) European Biological ControlLaboratory in Montferrier, France, and the Plant Pro-duction and Marketing BoardÐCitrus Division, IsraelCohen Institute for Biological Control in Bet Dagan,Israel. P. lounsburyi and P. humiliswere sent from theIsraeli colonies in 2009 and reared for two generationsonB. oleae at Kearney before the trials began. Rearingmethods were similar for both parasitoid species.Adult parasitoids were held in the Bug Dorm2 cages,provisioned with water and honey. Infested olivescontaining early third instar B. oleae were exposed toadult parasitoids in the holding cages. After a 2Ð3 dexposure period the olives were transferred to plastictrays, as described above, to rear the parasitized lar-vae.

Studies were conducted at a coastal site (UC Berke-ley, which is in the San Francisco Bay area and has acoastal Mediterranean climate) and two interior sites(Kearney as well as at the Lindcove Research andExtension Center, Lindcove, CA, which are �180 kminland and have hot, dry summers and colder wintersrelative to the coastal site). Unless otherwise noted, airtemperature data were taken from the California Ir-rigation Management Information System (CIMIS),which has stations in Berkeley, Kearney, and Lind-cove.Adult Fly Survival. The seasonal survival of adult

female ßies was measured in an olive orchard at Kear-ney. Beginning every 2 mo from February to October

468 ENVIRONMENTAL ENTOMOLOGY Vol. 42, no. 3

2005, adult ßies were placed in 10 cylindrical (25 by 45cm),organdy sleevecages(TufproNylonPaintStrain-ers, Warren Co., NC) that were hung on the east sideof olive tree canopies (two cages per tree). Ten femaleßies, 1Ð3 d old since adult eclosion, were released intoeach cage (100 per trial date). As a control, Þve iden-tical cages were established in the Kearney insectary(24 � 2�C, a photoperiod of 16:8 [L:D] h, 40Ð60% RH)at the start of each trial. Each cage was provisionedwith a 50% honey-water solution, provided in reser-voirs with cotton wicks, which were refreshed asneeded. The Þeld cages enclosed live olive brancheswithout fruit (all fruit were removed after bloom); theinsectary cages had extracted olive branches to pro-vide a similar substrate. Survival of the ßies was mon-itored weekly.Immature Fly Overwintering. Immature B. oleae

survival and development in overwintered olives weretested in Þeld-cage trials in a Lindcove olive orchard.In the Þrst trial, on 5 January 2007, 10 gravid femaleßies from the Kearney colony were placed in each of10 Tufpro sleeve cages that each enclosed olivebranches with 50Ð60 fruit per cage. No fruit was foundto naturally be infested by the ßy in this orchard (i.e.,all fruit within each cage were checked before eachtrial).After a3-wkexposureperiod,duringwhich foodand water were not provided, all tested adult ßies wereremoved. Subsets of 10Ð20 fruit were then randomlyselected from each cage to estimate initial ßy density,based on the number of oviposition stings on eachfruit. Subsequently, emergence of adult ßies in eachcage was monitored weekly until all ßies had emergedin the spring. The trial was repeated beginning on 25November 2008 and 2 December 2010, with the latertrial using 18 cage replicates. A similar trial was con-ducted, beginning on 25 November 2007, with 12 largecages (48 by 61 cm) that enclosed olive branches withmore (250Ð300) fruit and were inoculated with more(30) gravid female ßies.Immature Parasitoid Overwintering. Preliminary

observations suggested that immatures from P.lounsburyi and P. humilis eggs oviposited in Octoberwould be unable to complete development to thepupal stage before January, resulting in poor overwin-tering survival. To test this hypothesis, trials wereconducted in winter 2007Ð2008 and 2008Ð2009 in aKearney olive orchard. Trials consisted of the Tufprosleeve cages that each enclosed branches containing56Ð177 fruit (all fruit were free of natural infestationby the ßy). Approximately 3 wk before the scheduledrelease of parasitoids, each cage was inoculated with10 or 20 gravid female ßies, which remained for a 1 wkoviposition period and were then removed. The num-ber of fruit per cage was counted and a subsample offruit was checked, showing that the inoculation re-sulted in 1Ð3 B. oleae ovipositional scars (eggs or ovi-position attempts) per fruit. After the dissection ofsubsampled fruit indicated that the ßy larvae had de-veloped into early third instars, parasitoids wereadded to the cages.

In the Þrst trial, 20 mated female P. lounsburyi or P.humilis were released into each of 10 cage replicates,

from 4 October to 1 November 2007. In the secondtrial, 15 mated female P. lounsburyi or P. humiliswerereleased into each of 10 cage replicates, from 1 to 20October 2008. In the third and fourth trials, only P.humiliswas tested, with 15 females released into eachof 10 cages from 20 October to 12 November 2008, and20 females were released into each of 10 cages from 11November to 9 December 2008, respectively. Duringthese exposure periods, adult parasitoids were pro-vided with food (honey streaked on leaves and waterin vials with cotton wicks). After the exposure periods,all living wasps were removed from the cages andapproximately half of the fruit in each cage was col-lected, taken to the Kearney insectary, and held inplastic cups, under the insectary conditions describedpreviously, until ßies or wasps emerged. The other halfwas left in the cages to monitor emergence of para-sitoids or ßies in the Þeld. At the end of the trial, allunemergedpupariawere reconstituted inwater for1dand then dissected under a microscope to determinethe presence or absence of recognizable immatureparasitoid cadavers and pharate adults. Parasitism wasestimated based on the number of emerged and dis-sected wasps and ßies, while host density was esti-mated based on the total ßy puparia.Fly and Parasitoid Survival in the Soil. In fall, B.oleae commonly exit the fruit to pupate in the soilrather than inside the fruit; therefore, survival of pupalB. oleae (live or parasitized) in soil is critical for suc-cessful overwintering. To investigate ßy survivorshipunder these conditions, trials were conducted at acoastal site (an experimental farm at UC Berkeley)and an interior site (Kearney). For each trial, para-sitized and unparasitized B. oleae puparia were pre-pared following the same methodology for each loca-tion. Olive fruit were exposed to 100Ð200 female B.oleae in holding cages for up to 24 h, until each fruithad 3Ð5 oviposition scars. The infested fruit were thenheld for 910 d to allow the B. oleae to develop to earlythird instars, at which time the fruit were separatedinto two groups that were then exposed to either100Ð200 femaleP. humilisorP. lounsburyi for 24 h. Thenumber of parasitoids for each exposure varied, de-pending on their availability. After the exposure, ßypuparia were collected and inspected to remove anyunhealthy or dead pupae (e.g., dehydrated, diseased).The collected puparia were then randomly assigned totreatments.

Trials were initiated in October and repeated eachweek until the middle of December 2009 (a total of 10trials). At both sites, the tested puparia were separatedinto groups and placed in small (150 ml) plastic con-tainers Þlled with 50 g of soil, with the puparia buried1Ð2 cm below the soil surface. The sides and bottomof each container were punctured with small (�1 mmdiameter) holes to allow excess water to drain out, andthe container was covered with an organdy screen toprevent predators from entering. At each trial, allcontainers were held in a rectangular (10 by 50 by 120cm) wooden box with a metal mesh bottom; the boxwas Þlled with the same soil as in containers, andburied in the Þeld such that the soil level in the treat-

June 2013 WANG ET AL.: OVERWINTERING OF OLIVE FRUIT FLY AND PARASITOIDS 469

ment was the same as in the Þeld. A sandy loam soil wasused at each site to match the predominant soil type;at Kearney the soil was a premix (Robert Soria Truck-ing, Reedley, CA) and at UC Berkeley a sandy loamwas made using a 1:1 mix of sand and potting soil(Supersoil, Rod McLellan Company, Marysville, OH).An additional treatment imposed at each site was theinsectsÕ survival under two soil moisture regimes: nat-ural moisture, in which soil received natural rainfall,and controlled moisture, in which water was suppliedto the soil weekly to keep soil moisture between 60Ð90%, while rainfall was excluded by using a plasticcover held 2 m over the experimental arena.

Once each trial was initiated, the containers werechecked weekly for emergence of parasitoids or ßies.By the end of March when most ßies and wasps hademerged, all containers were collected and the soilwas examined for any remaining ßy puparia or para-sitized puparia, which were dissected and categorizedas either not emerged or dead. As a control, duringeach of the 10 trial periods, 30Ð40 (UC Berkeley) or50Ð200 (Kearney) puparia were kept in the insectaryroom until the emergence of ßies and parasitoids; thiscontrol was used to estimate adult ßy emergence,percentage parasitism, and emergence rates for eachtrial. There were Þve replicates for the natural mois-ture treatment and 9Ð15 replicates for the controlledmoisture treatment. During each trial, the number ofpuparia per replicate was similar, but varied amongtrial periods (range, 30Ð50) depending on the avail-ability of puparia. Air temperature, soil temperature,and humidity for each moisture treatment were re-corded using HOBO data loggers (Onset Corporation,Bourne, MA).Data Analysis. Data for all experiments are pre-

sented as means (�SE). Unless otherwise stated,treatment effects were compared using one-way, two-way, or three-way analysis of variance (ANOVA).Before analyses, data were transformed (e.g., arcsinesquare-root) as needed to normalize the variance.Analyses were performed using the statistical softwareof JMP (8.0, SAS Institute, Cary, NC).

For adult B. oleae Þeld survival, data were monthlypooled from all 10 cages to compare ßy survival be-tween the Þeld and insectary using Survival Analysis(log-rank test). If the overall log-rank test was signif-icant, a paired test of any two groups was made, withthe signiÞcance of paired comparisons adjusted to atreatment-wide level of � � 0.05 using the sequentialBonferroni adjustment. The cumulative percentagemortality S (t) over time (t) was described by a non-linear Weibull function: S (t) � 100 (one - exp (t/a)b),where a and b are Þtted parameters. The time at 50%survival was estimated by the Þtted model. In caseswhere some ßies were missing, the Þnal number wasbased on the observed live and dead ßies.

The percentage of successfully overwintering im-mature ßies was based on the estimated number ofeggs (at the start) and the number of emerged adults.Degree-day calculations were made from the date ofparent ßy release into cages to the date of Þrst adultoffspring emergence. Mean daily minimum and max-

imum Þeld temperatures from 1 December to 28 Feb-ruary were used to compare winter temperaturesamong years. The percentage of parasitoids survivingto the adult stage in the Þeld was estimated based onthe number of emerged wasps and the initial hostdensity and parasitism, as estimated from the labora-tory rearing of exposed fruit.

Overwintering survival of live and parasitized pu-pae buried in the soil was based on the initial numberof ßies and parasitoids (estimated by adult emergenceof the cohort in the insectary) and the number ofemerged ßies or parasitoids from Þeld cages at the endof the trial. The percent mortality in the Þeld wascorrected using SchneiderÐOrelliÕs formula, wherecorrected mortality � 100 (Þeld mortality � controlmortality)/(100 � control mortality). Control mor-tality � mortality under room conditions (Puntener1981). The relationship between cumulative percent-age of adult emergence and degree-days for both para-sitoids was analyzed using linear regression.

Previously, we compared the thermal performanceof the ßy and these two parasitoids, and the lower andupper developmental threshold from egg to adult forB. oleae, P. humilis, and P. lounsburyiwere 8 and 32�C,11 and 34�C, and 8.1 and 30�C, while thermal constantswere 380.8, 252.2, and 343.9 degree-days (DD), re-spectively (Wang et al. 2012). Degree-days were cal-culated based on these thresholds for each insect andusing the online double sine wave function program atthe University of California Integrated Pest Manage-ment Web site (http://www.ipm.ucdavis.edu).

Results

Adult Fly Survival. Adult female B. oleae caged atdifferent seasonal periods lived up to 231Ð343 and217Ð280 d under Þeld and insectary conditions, re-spectively (Table 1). The ßyÕs longevity in the insec-tary and the Þeld did not differ for trials in February(�2 � 1.38; df � 1; P � 0.239), April (�2 � 1.16; df �1; P � 0.282), June (�2 � 0.38; df � 1; P � 0.535), orAugust (�2 � 2.88; df � 1; P� 0.089), but in Octoberßies placed in the Þeld lived for a shorter period thanthose held in the insectary (�2 � 113.86; df � 1; P �0.001). Cumulative percentage mortality provided agood Þt to the nonlinear Weibull function, and pro-duced 50% mortality estimates that ranged from 98.7to 177.8 and 105.0Ð155.4 d for Þeld and laboratorytrials, respectively (Table 1). In the Þeld trials, periodsof higher mortality often coincided with periods ofextreme temperatures, especially low winter temper-atures (Fig. 1). Mean low weekly temperaturedropped to 3.4�C in January 2006, which coincidedwith the sudden death of 32 of 44 ßies and 60 of 88 ßiesover that cold period for trials initiated in August andOctober, respectively.Immature Fly Overwintering. B. oleae successfully

oviposited in blackened and overripe olive fruit pre-sented from late November through early January(Table 2). The number of eggs laid per fruit differedamong trial dates (F� 10.57; df � 3, 47; P� 0.001) asdid the percentage of ßies that successfully developed

470 ENVIRONMENTAL ENTOMOLOGY Vol. 42, no. 3

(F� 18.40; df � 3, 47; P� 0.001). Survival to the adultstage ranged from 0.23 to 8.50% and was positivelyrelated to mean daily minimum temperature (F �43.33; df � 3; r2 � 0.99; P� 0.002) but not mean dailymaximum temperature (F� 2.47; df � 3; r2 � 0.53; P�0.273). Minimum temperatures during the trial initi-ated on 7 January 2007 were signiÞcantly lower thanthose during the other trial periods (F � 11.98; df �3, 356; P � 0.001), with 21 d of daily minimum tem-perature below 0�C and a low of �5.7�C. Degree-dayaccumulation from oviposition to the Þrst emergenceof adult ßies ranged from 375 to 384 DD.Immature Parasitoid Overwintering. In 2007 and

2008, P. humilis and P. lounsburyi treatments had sim-ilar numbers of fruit per cage (2007: F� 0.36; df � 1,

18; P� 0.553 and 2008: F� 1.16; df � 1, 18; P� 0.2951)and host density per fruit (2007: F � 1.74; df � 1, 18;P � 0.203 and 2008: F � 0.98; df � 1, 18; P � 0.338)(Table 3). However, P. humilis parasitism levels werehigher than P. lounsburyi levels in both years (2007:F� 16.81; df � 1, 18;P� 0.001 and 2008:F� 27.17; df �1, 18; P� 0.001). Percentage of developed parasitoidswas not different between treatments (2007: F� 1.01,df � 1, 18, P � 0.331; 2008: F � 0.06, df � 1, 18, P �0.815) (Table 3). In 2007, a single P. lounsburyi com-pleted development to the adult stage, whereas no P.humilis emerged in the spring; in 2008 there was suc-cessful emergence of both parasitoid species, but�12% lived to the adult stage (Table 3). Dissectionsrevealed that P. humilis and P. lounsburyi commonlydeveloped to the pupal stage but died of dehydration(in the early trials) or disease (in the later trials). Forthe 2008 trials, conducted with only P. humilis, theinitial percentage parasitism was lower in late-Octo-ber (32.7%) and in mid-November (13.2%) trials, ascompared with those initiated in early-October (42.8and 64.5%), and no parasitoid successfully developedto the adult stage (Table 3).Fly and Parasitoid Survival in the Soil. Percentage

of insects surviving to the adult stage in the insectary(control) differed between the two insectaries (Kear-ney and UC Berkeley) and among insect species, av-eraging 56.9 � 5.0, 37.5 � 6.9, and 36.0 � 4.7% for B.oleaepupae,P. humilis, andP. lounsburyi, respectively,at Kearney, and 70.9 � 5.4, 51.2 � 7.9, and 37.8 � 7.4%by B. oleae puparia, P. humilis, and P. lounsburyi, re-spectively, at UC Berkeley. For this reason, survivaldata were corrected based on the survival of cohorts(for each trial, location, and species) in the insectariesat Kearney and UC Berkeley. Using the correctedoverwintering mortality data, there were signiÞcanteffects of location, trial date, and soil humidity treat-ment for species tested; and species, trial date, and soilhumidity for locations tested (Table 4). The pattern oftreatment effects was not consistent as there were noeffects of location for P. humilis, of trial date for B.oleae, or of soil moisture for either P. humilis or P.lounsburyi (Table 4). Mortality of B. oleae pupaeranged from 25 to 100% and was generally higher at UC

Table 1. Seasonal survival of adult female B. oleae in field or laboratory cages in Parlier, CA

TestStartdate

nMean � longevity

(in days)aMaximum

longevity (days)

Parameters of survivalmodela Estimated time to 50%

mortality (in days)ba b R2

Field 3 Feb. 77 174 � 7.8 287 31.65 1.68 0.850 177.87 April 76 162 � 5.8 280 27.84 2.32 0.879 166.69 June 78 106 � 7.5 231 17.61 1.67 0.945 98.718 Aug. 84 133 � 8.3 343 20.85 1.59 0.943 116.220 Oct. 89 98 � 3.5 273 17.75 2.58 0.819 107.8

Laboratory 3 Feb. 44 155 � 11.3 280 26.41 1.65 0.948 147.77 April 49 146 � 8.3 245 24.48 1.97 0.873 142.19 June 46 108 � 7.6 224 17.91 2.08 0.973 105.018 Aug. 50 125 � 7.0 217 20.75 2.22 0.947 123.220 Oct. 46 155 � 8.6 266 26.40 2.11 0.909 155.4

a Each trial began with 100 adult B. oleae; those ßies that disappeared (escaped or were not found) were not included in the data analysis.b The cumulative percentage mortality over time was Þtted to a nonlinear Weibull function and the time at 50% survival was estimated based

on the Þtted model.

Fig. 1. (A) Field maximum and minimum temperaturesand (B) survival of adult female B. oleae in Þeld cages set-upevery 2 mo from February to October 2005. Different lettersto the right of the legend key indicate a signiÞcant differenceof the survival curves among the different month releases(Survival Analysis; log-rank test; P � 0.05).

June 2013 WANG ET AL.: OVERWINTERING OF OLIVE FRUIT FLY AND PARASITOIDS 471

Berkeley than at Kearney (Fig. 2A and D). Mortalityof P. humilis increased during later trial dates, withnearly 100% mortality of pupal P. humilis in trialsinitiated after November (Fig. 2B and E). Mortality ofP. lounsburyi pupae was lower at UC Berkeley than atKearney and also increased in later trial dates (Fig. 2Cand F).

A higher percentage of B. oleae completed devel-opment than the parasitoids, with adult emergence �7d before the parasitoids in each location, and occur-ring from early- to mid-March. Those P. humilis and P.lounsburyi that completed development from the lasttrial dates on 18 December (UC Berkeley) and 17December (Kearney) emerged from mid- to lateMarch. Pooling all data for each location and species,cumulative emergence was positively related to cu-mulative degree-days (Fig. 3). The predicted degree-days at 50% emergence for P. humilis and P. lounsburyiwas 214.4 and 330.7 DD at UC Berkeley, respectively,and 216.7 and 303.3 DD at Kearney, respectively.

Discussion

Overwintering mortality is thought to be an impor-tant factor governing B. oleae population dynamics inEurope (Neuenschwander et al. 1981). In CaliforniaÕscoastal and interior olive growing regions, adult B.oleae have been caught in baited traps throughout theyear, although in relatively low numbers during thewinter and spring seasons (Yokoyama et al. 2006, Bur-rack et al. 2011). This current study shows thatB. oleaecan indeed survive and overwinter as an adult in Cal-iforniaÕs interior valley; female ßies held in Þeld cagesbeginning in October survived up to 273 d (until Junethe following year) when supplied with food and wa-

ter (Table 1). The sharpest declines in ßy numbersoccurred during the coldest part of winter (late Jan-uary), which was greater than that observed after highsummer temperatures that often exceeded 40�C fromlate July to early August (Fig. 1). Food, water, andtemperature are key factors inßuencing adult B. oleaesurvival (Wang et al. 2009a,b; Yokoyama 2012). Inolive orchards, natural food sources such as honeydewproduced by Saissetia oleae (Olivier) may sustain theßyÕs longevity (Wang et al. 2011a). Even with a foodsupply, adult ßies are vulnerable to CaliforniaÕs lowwinter temperatures, as showed in this study, and highsummer temperatures, as shown by Johnson et al.(2011). Although we did not test adult ßy survival inthe coastal site, adult overwintering survival is ex-pected to be greater in the coastal regions because ofmore mild winter and spring weather conditions.

We also showed that female B. oleae could ovipositon fruit in late fall and early winter and that the ßy eggssuccessfully developed into adults, although the per-centage survival of B. oleae eggs or larvae in overwin-tered fruit was also low (10%, Table 3) and immaturemortality was related to the daily lowest temperature.B. oleae was previously recovered from overwinteredfruit collected in coastal regions (Yokoyama et al.2006, Burrack et al. 2011), but not in the interiorvalleys. Avidov (1954) reported that adult B. oleae areinactive below 16.7�C. During our study at the Lind-cove site, there were only 6 d in January (2007) whenthe daily maximum temperature was higher than16.7�C. Thus, the adult ßy survived during the colddays and oviposited during the few warm days orperiods of those days.

Previous researchers have shown that the ßies over-winter as adults or immatures in fruit (Kapatos and

Table 2. Overwintering survival and spring emergence of immature B. oleae in field cage trials initiated in Jan., Nov., and Dec. inLindcove, CA

Start date Flies/cage n Fruit: ßy Eggs/fruitb% ßies

developedbMaximumT (�C)b

MinimumT (�C)b

First ßyemergence

Degreedaysb

5 Jan. 2007 10 10 5.1:1 1.17 � 0.42a 0.23 � 0.21a 15.4 � 0.5a 1.2 � 0.50a 31 Mar. 37525 Nov. 2007a 30 12 9.3:1 2.55 � 0.30b 4.61 � 1.85b 14.7 � 0.3a 2.8 � 0.32b 19 Mar. 38925 Nov. 2008 10 10 6.1:1 3.51 � 0.35c 6.52 � 1.53bc 13.8 � 0.4a 3.3 � 0.34b 30 Mar. 3852 Dec. 2010 10 18 5.0:1 1.54 � 0.18ab 8.50 � 1.76c 14.5 � 0.4a 4.0 � 0.33b 28 Mar. 384

a Large cages were used in this trial.b Values are mean � SE, and temp refers to mean daily max and min. temp (T) from 1 Dec. to 28 Feb. of each winter period. Degree day

was calculated based on temperatures from the start date to the Þrst adult ßy emergence. Within each column, different letters after treatmentmeans indicate a signiÞcant difference (P � 0.05).

Table 3. Survival to the adult stage (mean � SE) for caged P. humilis and P. lounsburyi during Oct., Nov., and Dec. in 2007 and2008 in Parlier, CA

Year SpeciesWasps/

cageDate wasps

cagedDate wasps

removedFruit/cage

B. oleaedensity/fruit

PercentageParasitism

Percentage successfuldevelopment

2007 P. humilis 20 4 Oct. 1 Nov. 117.4 � 4.0a 1.23 � 0.16a 42.8 � 3.3a 0aP. lounsburyi 20 4 Oct. 1 Nov. 110.8 � 10.2a 1.75 � 0.35a 10.2 � 4.0b 4.3 � 4.0a

2008 P. humilis 15 1 Oct. 20 Oct. 69.5 � 5.0a 1.11 � 0.26a 64.5 � 9.3a 5.6 � 3.3aP. lounsburyi 15 1 Oct. 20 Oct. 76.3 � 3.8a 0.77 � 0.23a 7.8 � 3.8b 11.9 � 6.4a

2008 P. humilis 15 20 Oct. 12 Nov. 56.0 � 4.2 1.05 � 0.18 32.7 � 5.7 0P. humilis 15 11 Nov. 9 Dec. 57.4 � 5.9 0.86 � 0.10 13.2 � 6.8 0

For each year and parameter measured, different letters after treatment means indicate a signiÞcant difference (P � 0.05).

472 ENVIRONMENTAL ENTOMOLOGY Vol. 42, no. 3

Fletcher 1984) or as pupae in the soil (Michelakis1980, Neuenschwander et al. 1981). We showed thatßy pupae, buried in soil from fall (October) to winter(December), survived and emerged as adults in spring(March) in both the coastal and interior sites (Fig. 2).Combined with the studies of adult and immaturestages, this suggests that B. oleae may overwinter inCalifornia as adults, immatures in the fruit, or pupae inthe soil. We suspect that B. oleae that reach the pu-parial stage in late fall are the more likely overwin-tering stage in olive growing regions with colder win-ter temperatures. The phenology of B. oleae inCalifornia seems to be similar to that in Greece, where

adult ßies also emerge in spring and attack olivesremaining on the trees from the previous season(Kapatos and Fletcher 1984).

Low temperature and high soil moisture have beensuggested asB. oleaemortality factors (Neuenschwan-der et al. 1981) and, in this study, pupal mortality wasinßuenced by winter temperature and soil moisture(Table4;Fig. 2). InGreece,Michelakis (1980) showedthat the emergence of adult ßies, buried in the soil,ranged from 0 to 20% when recorded from the coldermountain regions to the mild lowlands, respectively.In Portugal, Goncalves et al. (2012) estimated thatmortality of buried puparia reached 98.5% in northÐeastern Portugal. Pupal mortality will also depend onother conditions such as disease and predation(Neuenschwander et al. 1981, Orsini et al. 2007, Gon-calves et al. 2012). In this study, predators were ex-cluded and we suspect most mortality resulted fromdehydration before the winter rains (October andNovember, 1.45and0.34 inches forBerkeleyandKear-ney sites, respectively) and from diseases in wet soilsduring winter rainfall (December to March, 14.26 and7.27 inches for UC Berkeley and Kearney sites, re-spectively).

Concurrent to our investigation of B. oleae over-wintering survival and development, we conductedsimilar studies with the two solitary, endoparasiticbraconids. Previously, we showed that P. humilis andP. lounsburyi can attack B. oleae inside fruit in late fallor early spring (Wang et al. 2011b). Here, we showedthat these parasitoid species can overwinter as imma-ture stages and reach the adult stage in early spring onoverwintered hosts that either remains inside fruit onthe tree or inside ßy larvae that drop from the fruit topupate in the soil. Parasitoid mortality during thisperiod was high (Table 4; Fig. 2).

Table 4. Results of three-way ANOVA testing the effects oflocation, burial date, and soil humidity on overwintering mortalityof B. oleae puparia, P. humilis, and P. lounsburyi or the effects ofdifferent insect species, burial date, and soil humidity on the over-wintering mortality at Berkeley or Parlier, CA

Parameter Factors df F P

P. humilis Location 1 2.47 0.1170Burial date (B) 9 4.27 �0.0001Humidity (RH) 1 3.22 0.0735B � RH 9 0.36 0.9528

P. lounsburyi Location 1 17.75 �0.0001Burial date (B) 9 2.39 0.0130Humidity (RH) 1 2.53 0.1126B � RH 9 0.72 0.6905

B. oleae Location 1 37.81 �0.0001Burial date (B) 9 1.39 0.1879Humidity (RH) 1 11.14 0.0009B � RH 9 0.98 0.4577

Berkeley Species 2 16.21 �0.0001Burial date (B) 9 2.94 0.0020Humidity (RH) 1 11.30 0.0008B � RH 9 1.43 0.1748

Parlier Species 2 1.08 0.3405Burial date (B) 9 5.37 �0.0001Humidity (RH) 1 7.29 0.0071B x RH 9 1.07 0.3824

Fig. 2. Corrected overwintering mortality of (A and D) B. oleae, (B and E) P. humilis, and (C and F) P. lounsburyi pupaeburied in the soil from the middle October to the middle of December 2009 (once per week) at a coastal site in Berkeley(A, B, and C) and at an interior site in Parlier (D, E, and F), CA. Additionally, at each site the amount of soil moisture inthe burial arena was either natural (i.e., the soil receiving natural rainfall) or controlled soil humility in which the arena wassprayed with water on a weekly basis to maintain the soil humidity at around 60Ð90% and the arena was covered for protectionfrom rains.

June 2013 WANG ET AL.: OVERWINTERING OF OLIVE FRUIT FLY AND PARASITOIDS 473

Overwintering mortality of immature P. humilis inßy puparia was affected by burial date (that corre-sponds with temperature) and immature P. lounsburyiwere affected by both burial date and location; thelater species had better survival at the coastal site thanthe interior site (Table 4; Fig. 2). Low temperaturekills some parasitoid species by causing physical ormetabolic injury (Hance et al. 2007). Continual ex-posure of P. humilis eggs or larvae to low temperature(�10�C) resulted in higher mortality than that expe-rienced by its pupae (Daane et al. 2013). In laboratorystudies, P. humilis appears to be less cold tolerant thanP. lounsburyi or P. ponerophaga (Daane et al. 2013).The Þeld study reported herein supports this obser-vation: whereas winter morality was extremely highfor both parasitoid species, P. humilis survival waslower than P. lounsburyi survival at the coastal site(Fig. 2B and C).

Our previously developed degree-day models forB.oleae, P. humilis, and P. lounsburyi (Wang et al. 2012a)provided a relatively accurate prediction of the over-wintering development of these insects. For P. humilisand P. lounsburyi, overwintering development waslinked to temperature in each year of the study, sug-gesting these parasitoids do not have a winter dia-pause; species that do not diapause are often lesscold-tolerant (Hance et al. 2007). While some fruit ßyparasitoid species from temperate and tropical regionsin Asia and the Americas are known to enter diapause(Aluja et al. 1998, Carvalho 2005), none from subtrop-ical Africa have been reported. Without a diapause itis critical for adult P. humilis and P. lounsburyi to Þnd

hosts soon after they emerge in early spring. In Cal-ifornia, the lack of alternative hosts, such as Medßy,may require even better synchronization among theßy, introduced parasitoids, and environmental condi-tions. One possible survival mechanism would be along adult survival period, and a previous studyshowed that adult P. humilis could survive for up to150 d during the overwintering period, when providedaccess to food, and surviving females could still suc-cessfully oviposit (Wang et al. 2011a). The relativelylong-lived adult parasitoids and the availability ofadult parasitoid food sources, such as honeydew, areprobably necessary for successful establishment ofthese species in new geographic ranges where noalternative hosts are available.

This work suggests that among the desired traits forB. oleae parasitoids is tolerance to overwintering tem-perature. Previous studies elucidated other inherentdifÞculties of classical biological control of B. oleae.First, domesticated olives are substantially larger thanwild olives, which limit the effectiveness of larvalparasitoids with short-ovipositors adapted to smallwild olives (Wang et al. 2009c,d). Second, domesti-cated olives have a more uniform ripening period,reducing the availability of suitable fruit as B. oleaehosts during some periods in the year (Copeland et al.2004). In California, although different olive cultivarsßower and mature their fruit at slightly different times(Burrack and Zalom 2008), a postßowering periodexists in spring and early summer when the previouscrop of olive fruit has disappeared and the new cropof olive fruit are not mature enough for B. oleae de-

Fig. 3. A positive relationship was found between degree-days (from egg to adult emergence) and the percentagecumulative emergence for Psyttalia humilis (E, F) and P. lounsburyi (‚, Œ) at a coastal site in Berkeley (A and B) and aninterior valley site in Parlier (C and D), CA, when parasitized B. oleae puparia were buried in the soil on different trial datesfrom October through December (see Fig. 2); regression slopes for each Þgure are: (A) y � �52.9 � 0.48x, r2 � 0.27, P �0.001; (B) y � �149.4 � 0.492x, r2 � 0.48, P � 0.001; (C) y � �85.6 � 0.41x, r2 � 0.38, P � 0.001; (D) y � �68.3 � 0.39x,r2 � 0.17, P � 0.001.

474 ENVIRONMENTAL ENTOMOLOGY Vol. 42, no. 3

velopment (Yokoyama et al. 2012). In addition, in theßyÕs native range other tephritid species are presentthat serve as alternative hosts for many of the parasi-toid species that attack B. oleae (Copeland et al. 2004,Wharton et al. 2000). Here, we have looked closely atonly two of the common B. oleae parasitoid species;there are possibly other species outside Africa thatmay be better adapted for overwintering in the ßyÕsexpanded range, although they have not yet beenfound or evaluated (Hoelmer et al. 2011).

Acknowledgments

We thank Brianne Crabtree, John Hutchins (University ofCalifornia, Berkeley), and Martha Gerik (University of Cal-ifornia, Riverside) for assistance. Funds were provided by theCalifornia Specialty Crop Block Grant (administered by theCalifornia Department of Food and Agriculture, with fund-ing from USDA), the California Olive Committee, and theUSDA CSREES Special Grants Program: Pest ManagementAlternatives. Voucher specimens are deposited at the UCBerkeley Essig Museum.

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Received 17 October 2012; accepted 11 April 2013.

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