Comparison of two laboratory cultures of Psyttalia concolor (Hymenoptera: Braconidae), as a parasitoid of the olive fruit fly

Biological Control 39 (2006) 248–255 www.elsevier.com/locate/ybcon

Comparison of two laboratory cultures of Psyttalia concolor (Hymenoptera: Braconidae), as a parasitoid of the olive fruit Xy Karen R. Sime a,¤, Kent M. Daane a, Russell H. Messing b, Marshall W. Johnson c a

Division of Organisms and Environment and Center for Biological Control, University of California, Berkeley, CA 94720-3114, USA b University of Hawaii, Kauai Agricultural Research Center, 7370 Kuamoo Road, Kapaa, Kauai, HI 96746, USA c Department of Entomology, University of California,Riverside, CA 92521, USA Received 2 March 2006; accepted 14 June 2006 Available online 27 June 2006

Abstract Life-history trials were conducted in the laboratory on two cultures of Psyttalia concolor (Szépligeti) (Hymenoptera: Braconidae) reared on olive fruit Xy, Bactrocera oleae (Rossi) (Diptera: Tephritidae). The tested cultures (A and B) had similar histories except that Culture A was maintained in Kenya for four years. Results showed that parasitoids from both cultures preferentially searched on olive fruit containing 8 to 10-day-old (second and third instar) olive Xies, and reproduced most successfully when third-instar Xy larvae were available. The mean longevity of adult female P. concolor was a negative function of temperature. Females from Culture B lived signiWcantly longer at 15, 22, 25, and 32 °C than females from Culture A. For both cultures, adult female longevity was signiWcantly longer when the parasitoids were provided with honey than when provided water alone, or nothing; the presence of hosts signiWcantly reduced longevity, suggesting an energetic cost for reproduction. The parasitoids produced an average of 28.7 § 4.1 and 22.2 § 5.1 oVspring per female in Cultures A and B, respectively. The results are discussed with respect to use of biological control agents held under diVerent rearing conditions, and the potential of P. concolor for use as a biological control agent for olive fruit Xy in California. © 2006 Elsevier Inc. All rights reserved. Keywords: Psyttalia concolor; Braconidae; Bactrocera oleae; Tephritidae; Olea; Biological control; Parasitoid biology; Olive

1. Introduction The olive fruit Xy, Bactrocera oleae (Rossi) (Diptera: Tephritidae), has long been a pest of olives in the Mediterranean basin (Clausen, 1978; Tzanakakis, 2003; White and Elson-Harris, 1992). It was discovered in southern California in 1998 and within four years had spread nearly throughout the state, posing a serious threat to the olive industry (Rice et al., 2003). Current research eVorts emphasize the development of sustainable management practices. Insecticidal baits are currently the principal means of control, but are limited in their eVectiveness. Infested trees in suburban and rural landscaping act as reservoirs for reinvasion (Collier and Van Steenwyk, 2003), and, furthermore,

*

Corresponding author. Fax: +1 510 643 5438. E-mail address: [email protected] (K.R. Sime).

1049-9644/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2006.06.007

the successful biological controls that have been established for scale pests in California olives (Daane et al., 2005) might be disrupted by insecticides. As no natural enemies exist in California that can eVectively suppress olive Xy, a classical biological control program was initiated in 2002. EVorts to identify useful natural enemies have to date included foreign exploration in Africa and Central Asia, and evaluation of parasitoids used to control tephritid pests elsewhere (Hoelmer et al., 2004; Sime et al., 2006). Among the olive Xy parasitoids readily available in culture is Psyttalia concolor (Szépligeti) (Hymenoptera: Braconidae). Recently, this species was released in California on a limited basis (Collier and Van Steenwyk, 2003), but it has not yet been approved for widespread, open-Weld release, pending evaluation of its potential eVects on nontarget species. Although P. concolor has been reared from numerous tephritid species in the laboratory and in agricultural settings (Wharton and Gilstrap, 1983), only two

K.R. Sime et al. / Biological Control 39 (2006) 248–255

species (the olive Xy and the Mediterranean fruit Xy (MedXy), Ceratitis capitata (Wiedemann)) are known as typical hosts in its native range. In parts of northern and eastern Africa, P. concolor is regularly reared from olive Xy in both wild and cultivated olives (Clausen, 1978; Copeland et al., 2004; El-Heneidy et al., 2001; Gaouar and Debouzie, 1991), and it is also frequently reared from MedXy in Kenya (Wharton et al., 2000) and North Africa (Narayanan and Chawla, 1962). European programs for olive Xy biological control have focused on P. concolor to the exclusion of almost all other parasitoid species, especially after an eYcient mass-rearing method (using MedXy in artiWcial diet) for P. concolor was developed in the 1950s (Greathead, 1976). Shortly after it was Wrst identiWed in material collected in Tunisia in 1910, P. concolor was introduced to Italy and France, and in subsequent decades was released widely in the olive-growing regions of southern Europe. Few of these introductions resulted in establishment, however, and where it has established, inundative releases are required to boost parasitism rates (Clausen, 1978; Greathead, 1976). Wharton (1989) has argued that the nearly century-long reliance on P. concolor has had more to do with the ease of collecting and rearing this species, compared to other olive Xy parasitoids, than with its proven eVectiveness as a control agent. He further argues that this “misguided” emphasis on P. concolor has come at the expense of investigating the potential of other parasitoid species. Despite this history, we considered P. concolor an important species to screen for use in California’s biological control program against the olive Xy. Several considerations suggest that P. concolor merits further investigation. First, mass cultures of P. concolor are maintained on MedXy, because this host is easily reared on artiWcial diets. Changes in P. concolor mating and oviposition behavior have been noted in cultures maintained for many generations, however, and it has been suggested that these and other traits associated with the ease of rearing may diminish P. concolor eVectiveness in the Weld (Kimani-Njogu et al., 2001), especially as a parasitoid of the olive Xy (i.e., a novel host outside of the culture). ModiWcations of culture protocols, such as increasing the addition of wild stock and rearing on olive Xy rather than MedXy, may thus improve the performance of P. concolor in an olive Xy program. These modiWcations are now possible with improvements in rearing methods. (In California, rearing on MedXy is impossible, because MedXy is not considered to have established and it cannot be legally imported into the continental USA (Headrick and Goeden, 1996).) Second, P. concolor is a widespread species: its native range spans most of arid northern and eastern Africa (Kimani-Njogu et al., 2001; Wharton and Gilstrap, 1983). It likely comprises several cryptic species or genetically diVerentiated populations, and given the diversity of habitats and climates encompassed by this distribution, there may exist races adapted to particular environments or hosts (Diehl and Bush, 1984; Hopper et al., 1993; Kimani-Njogu et al., 2001; Wharton and

249

Gilstrap, 1983). Despite the mixed results in biological control programs, no attempt has yet been made to compare the biology of P. concolor from diVerent regions speciWcally to determine whether some populations are diVerentially adapted to olive Xy or to MedXy, or whether some populations fare better in certain climates. Finally, the biology of P. concolor is known almost entirely from studies conducted using MedXy in artiWcial diet as the host (Avilla and Albajes, 1983; Biliotti and Delanoue, 1959; Canale and Raspi, 2000; Canard et al., 1979; Loni, 1997; Stavraki-Paulopoulou, 1966; Wang and Messing, 2004). The identiWcation of diVerences in its biology when attacking olive Xy larvae reared in whole fruit could lead to improvement of insectary and Weld-release protocols. The goals of this study were to investigate the biology of P. concolor when using olive Xy as the host, in olive fruit, and to determine whether intra-speciWc variation exists in biological traits relevant to its performance in the insectary and in the Weld. To begin our investigation of intra-speciWc variation in P. concolor, we worked with two longstanding laboratory cultures of this species. Although both cultures came from stock maintained on MedXy, we switched both to olive Xy for approximately ten generations prior to the beginning of this study, and conducted all experiments with olive Xy (reared in picked fruit) as host. To investigate host adaptation, we compared the oviposition behavior and fecundity of females from the two colonies. To investigate their hardiness in diVerent conditions, we compared female longevity at diVerent temperatures and given diVerent provisions. The short-term goal of these studies was to determine whether either or both of the cultures would provide improved stock for innoculative release against olive Xy in California. In the long term, however, determining whether there exists genetic variation for host compatibility or climate tolerance in P. concolor will help direct foreign exploration for biological control of both the olive Xy and the MedXy, and will help improve mass-production techniques. 2. Materials and methods 2.1. Sources of insects and plants, and culture maintenance Laboratory cultures of olive Xy were derived from infested olives collected near Davis, California (Yolo County). Flies were reared on olive fruit following the methods set forth by Tzanakakis (1989, 2003). Because the Xies do not develop on immature fruit less than two months old, and olives picked when fully ripe tend to rot before the Xy larvae (or their parasitoids) complete development, we used a variety of olive cultivars (mostly Manzanillo, Sevillano, and Mission) with varying periods of ripening. These cultivars could be collected at diVerent times across a long section of the state (Riverside, Kern, Tulare, Fresno, and Yolo Counties), thereby providing fruit of an acceptable quality for 9 to 10 months out of the year. Olives held in cold storage were used for the remaining period.

250

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Olives were exposed to adult Xies in an ovipositional chamber (45 £ 45 £ 45 cm wooden cage with organdy sides and a glass top) that was kept in a temperature-controlled room (22 § 2 °C, 16:8 (L:D)h, 40% RH) at the University of California Berkeley Insectary and Quarantine Facility (Berkeley I&Q). The adult Xies had free access to water and a mixture of honey and a dry yeast extract (2:1 by volume) (FisherBiotech, Fairlawn, New Jersey). Olives were left in the cage for 1–2 d or until each had 5–10 oviposition marks. Infested olives were then transferred to plastic boxes (36 £ 18 £ 10 cm) with wire-mesh tops. To reduce mold growth, olives were placed in the box no more than 2–3 layers deep and were raised about 2 cm oV the bottom of the container by a metal grid. Under these conditions, the mature larvae exited the fruit and pupated on the box bottom after 10–14 d. Puparia were collected and transferred to the ovipositional chamber to emerge as adults and repeat the process. The two P. concolor laboratory cultures used in this study originated with material Weld-collected in North Africa in the 1970s and then maintained for decades in Italy (University of Pisa), where they were reared on MedXy (Canale, 1998). This initial “Italian” culture is the same culture referred to by Kimani-Njogu (2001), Loni (1997), and Raspi and Canale (2000). Our Wrst culture, referred to hereafter as “Culture A”, was sent from the Italian culture to Kenya (International Center of Insect Physiology and Ecology (ICIPE), Nairobi) in 1998, from Kenya to Hawaii (Kauai Agricultural Research Center) in 2002, and then from Hawaii to Berkeley I&Q in 2003. The second culture, referred to hereafter as “Culture B”, was sent from the Italian culture to Hawaii (Kauai Agricultural Research Center) in 1999 and then from Hawaii to Berkeley I&Q in 2003. The cultures thus had similar backgrounds before arriving at Berkeley I&Q for our studies. The important diVerence is that Culture A left Italy in 1998 and was maintained in Kenya for four years. Otherwise, these colonies should represent the same population. Culture techniques were similar. The only diVerence we are aware of is that in Kenya (i.e., Culture A) the culture was maintained at a slightly higher temperature (27–28 °C) (Mohamed et al., 2003) than in Hawaii (20–24 °C) (Wang and Messing, 2002). The adult parasitoids were held in 45 £ 45 £ 45 cm3 cages (as described for the olive Xy) that were freely provisioned with water and a honey–water solution (50% by volume) and kept in a temperature-controlled room (22 § 2 °C, partial natural light augmented by 16:8 (L:D)h, 40% RH). Previous reports describe P. concolor (reared on C. capitata) as a koinobiont that favors third (last) instar larvae for oviposition, with the larva completing its development in the host’s puparium (Biliotti and Delanoue, 1959; Canale, 1998; Wang and Messing, 2004). Olives infested 6–10 d earlier and thus containing a mixture of second- and thirdinstar Xy larvae were exposed to parasitoids for 1–3 d, depending on parasitoid density. The inoculated material was then transferred to plastic boxes as described above. The Xy larvae dropped to the bottom of these containers to

pupate, at which time the puparia were collected and transferred to transparent plastic Petri dishes (9-cm diam) that were monitored daily for the emergence of adult Xies and parasitoids. Experiments described below were conducted beginning in August 2004, thus over a year (t10 generations) after the cultures were brought to Berkeley I&Q and switched to rearing on olive Xy. 2.2. Host stages used for oviposition Host-stage preference and reproductive success on diVerent olive Xy development stages were examined in choice tests. To produce an age series of olive Xies, fresh olives were exposed to adult Xies for 8 h every 2 d and then held at 25 § 1 °C. The olives were presented to the parasitoids when the Xy larvae were 2, 4, 6, 8, 10, and 12-d-old. A subsample of olives from each set was dissected shortly before the test to determine which larval stages were present. Under these conditions, 2-d-old olives contained eggs and, rarely (<5%), Wrst instars; 4-d-old olives contained Wrst instars; 6-d-old olives contained second instars; 8-d-old olives contained second and young third instars; 10-d-old olives contained third instars; and 12-d-old olives contained mature third instars and were accompanied by prepupal larvae (emerging from the fruit) and occasionally puparia. For each replicate, four female parasitoids were held for 24 h in an ovipositional chamber (a translucent white plastic cylinder 13 cm deep £20 cm diam, with a Wne mesh top) containing 24 olives, of which four each were infested by one of the six olive Xy age classes. The olives were placed in the bottom of the container, with the olives of each olive Xy age class grouped in open Petri dishes (5-cm diam). During the Wrst 7– 8 h of the exposure period, ten 5-s observations were made, at approximately 40-min intervals, of activity within the containers. The age class of olives contacted by parasitoids was recorded. After the parasitoids were removed from the ovipositional chamber, the olives were held at 25 § 1 °C to rear either adult parasitoids or olive Xies. There were 7 replicates for Culture A and 10 replicates for Culture B. 2.3. Adult longevity at various temperatures Adult male and female longevities were measured at six temperatures (15.2 § 0.3, 21.9 § 0.2, 24.8 § 0.4, 27.8 § 0.2, 30.1 § 0.2, and 32.0 § 0.5 °C). Newly emerged parasitoids were placed singly in glass vials (5-cm long £ 1-cm diam, with a mesh lid), provisioned with a streak of honey–water (50% solution by volume), and randomly assigned to a temperature cabinet. The parasitoids were checked daily for mortality. The honey–water was replenished every 2–3 d or as needed. From each culture, 10 male and 10 female parasitoids were tested at each temperature. 2.4. Adult longevity given diVerent provisions Female longevity was compared among Wve treatments with access to (1) olives containing hosts, honey–water

K.R. Sime et al. / Biological Control 39 (2006) 248–255

(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 with water and honey–water, and held for 2 d to mate. The females were then randomly assigned to one of the Wve treatments, with each parasitoid isolated in a small plastic container (15 cm diam £ 6 cm deep) with a hole (7 cm diam) cut in the lid and covered with nylon mesh for ventilation. The olives (four per container) were replaced every other day. Where olives with hosts were oVered, the Xy larvae were at a suitable stage for parasitoid oviposition (mostly third instars). Each of the four olives had 5–10 oviposition marks, and therefore some 20–40 larvae were available over each 2 d interval. Honey-water, streaked along the sides of the container, and distilled water, in a soaked cotton wick, were freely available. Parasitoids were checked daily for mortality. All treatments were kept in a temperature-controlled room (22 § 2 °C, 40% RH, 16:8 (L:D) h supplemented by natural daylight). Each treatment was replicated 10 times for each culture.

A 45 Parasitoid encounters (%)

40

2.6. Statistics Results are presented as means per treatment (§SE). Treatment eVects were analyzed using analysis of variance (ANOVA), with treatment means separated using Tukey’s HSD test (three or more treatments) or t tests (two-way comparisons). 3. Results and discussion 3.1. Host stages used for oviposition Host-stage preference, as measured by females searching on fruit (Fig. 1A) and by reared parasitoid oVspring (Fig. 1B), was a positive function of olive Xy larval age (age class) for Cultures A and B. Within each olive Xy age class, there were no signiWcant diVerences between cultures for either the percentage encounters or oVspring reared (Table 1). For this reason, data from the cultures were combined to better assess host-stage preference. Olives containing all age classes of olive Xy stages were examined by P. concolor, as suggested by searching behaviors (Fig. 2A), and contained acceptable host stages from which parasitoids were reared (Fig. 2B). SigniWcantly more olives containing 8- and 10-d-old olive Xy larvae were searched than fruit containing 2 and 4-day-old

35 30 25 20 15 10 5 0 0

2

4

6

8

10

12

14

8

10

12

14

Parasitoid offspring (%)

B 70

2.5. Lifetime fecundity To determine lifetime reproductive potential, the infested olives that were collected every other day in the experiment described previously (Section 2.4) were held in plastic cups for the emergence of adult Xies or parasitoids. The number and sex of the emerging oVspring were recorded.

251

60 50 Colony A Colony B

40 30 20 10 0 0

2

4

6

Olive fly larva age (in days)

Fig. 1. Psyttalia concolor host-stage preference was a positive function of olive fruit Xy larval age class as measured by (A) the percentage contact encounters, for both Culture A (y D 0.306 + 2.334x, r2 D 0.188, F D 10.49, df D 1,40, P D 0.002) and Culture B (y D 4.912 + 1.6794x, r2 D 0.069, F D 5.419, df D 1,58, P D 0.023); and (B) the percentage oVspring reared, for both Culture A (y D ¡13.19 + 4.256x, r2 D 0.253, F D 14.89, df D 1,40, P < 0.001) and Culture B (y D ¡8.072 + 3.5346x, r2 D 0.159, F D 12.23, df D 1,58, P < 0.001).

Table 1 For each olive Xy age class, there was no signiWcant diVerence between Psyttalia concolor cultures A and B in percentage encounters (see Fig. 1A) or oVspring (see Fig. 1B) Olive Xy age class

Percentage encounters t-statistic

P-value

t-statistic

P-value

2-d-old 4-d-old 6-d-old 8-d-old 10-d-old 12-d-old

0.013 0.808 1.151 0.767 0.760 1.533

0.236 0.989 0.267 0.455 0.459 0.146

1.226 0.823 0.332 0.451 0.494 0.553

0.239 0.423 0.744 0.659 0.628 0.588

No. oVspring

Two-sample t-test of Culture A and B for each olive Xy age class, df D 15 for each.

larvae. Oviposition success was highest in 10-day-old olive Xy larvae, and signiWcantly more parasitoids were reared from the 10 and 12 day age classes than from the 2 and 4 day age classes (Fig. 2B). The preference for older olive Xies was most evident in Culture A, where no Xies were reared from the 2 and 4 day age classes (Fig. 1B).

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K.R. Sime et al. / Biological Control 39 (2006) 248–255

A

40 b

Parasitoid encounters (%)

35

b

30

ab 25 ab

20 15 a 10

a

5 0 2

B

4

6

8

60

10

12

c

Parasitoid offspring (%)

50 b 40 30 20 10

a

ab

ab

6

8

a

0 2

4

10

12

Olive fly larva age (in days)

Fig. 2. Psyttalia concolor host-stage preference was signiWcantly diVerent among olives containing 2 to 12-day-old olive Xy larvae as measured by (A) the percentage contact and probing encounters (F D 5.490, df D 5, 96, P < 0.001) and (B) the percentage oVspring reared (F D 7.917, df D 5, 96, P < 0.001). Data are combined for cultures A and B. In each graph, diVerent letters above each bar indicate signiWcant diVerences in treatment means (Tukey’s pairwise comparison, P < 0.05).

These results indicate that there is some level of host habitat selection. Females searched signiWcantly more on olives containing second- and third-instar Xy larvae (8 and 10 day age classes) than on olives containing eggs and Wrst and second instars (2, 4, and 6 day age classes) or mature third instars and pupae (12-day age class). We also observed a discrepancy between preference measured in terms of female choice encounters and their success in reproducing on diVerent larval instars. The greatest level of parasitoid and host encounters was on olives in the 8-day age class (2A), but the greatest level of oviposition success was in the 10-day age class (2B). One explanation for this discrepancy may lie in the relatively short ovipositor ( » 2 mm) of P. concolor females (Wharton and Gilstrap, 1983), in that they cannot reach larvae that have burrowed deep into the olive. In this study, olive dissections revealed that second instars typically fed deep in the olive, near the pit, well out of reach of the parasitoid’s ovipositor. Thirdinstar larvae varied in their feeding depth and were often found close to the surface. Wild olives are much smaller than domestic olives (1 cm diam or less, compared to

2–3 cm) (Bartolini and Petruccelli, 2002), so these diVerences in feeding habits would have less eVect on the reproductive success of P. concolor in its native environment, using hosts in wild olives. Consequently, this species may not have evolved the ability to discriminate between olives infested with second and third instars. In contrast, no such discrepancy between oviposition preference and success was found for two Diachasmimorpha (Braconidae) species (D. kraussii (Fullaway) and D. longicaudata (Ashmead)) tested under identical experimental conditions, although both preferred second and young third instar larvae (Sime et al., 2006). The Diachasmimorpha species have longer ovipositors, 0.5–1 cm. It is also possible that the reduction in host encounters on olives in the 10- and 12-day age class resulted from a reduction in olive quality or a change in volatiles from the picked fruit. However, we did not observe any outward changes in olive color or texture during the 12-day development period. These results agree with most accounts of P. concolor that indicate greater reproductive success (on Mediterranean fruit Xy) when older hosts are attacked (Biliotti and Delanoue, 1959; Wang and Messing, 2004; Wharton et al., 2000). These studies have not compared female choice with reproductive success, however. In the laboratory, P. concolor is readily reared from MedXy exposed as early as the Wrst and second instar (Raspi and Canale, 2000). This work was conducted using hosts in artiWcial diet, where the ability of the ovipositor to reach the host is apparently irrelevant. Based on this Wnding, and the observation that P. concolor is capable of parasitizing Wrst-instar olive Xy (which we found as well, though in very low numbers), Raspi and Canale (2000) recommended that P. concolor be released in the Weld when olive Xy populations are mostly in the Wrst and second instar. Our results contradict this recommendation, indicating that in cultivated olives P. concolor will have its greatest reproductive success when the host population consists mostly of third instars. 3.2. Adult longevity at various temperatures Adult longevity was a negative function of temperature for each combination of culture and gender (Fig. 3). The eVect of temperature on adult longevity was the only consistent diVerence found between the cultures. At all temperatures except 28 °C, adult females from Culture A lived for shorter periods than females from Culture B. The diVerences were not as pronounced for adult males, where only at temperatures of 22, 25, and 28 °C did Culture A males live for signiWcantly shorter periods than Culture B males (30 °C signiWcant at P < 0.1). DiVerences in climate tolerance should be considered in the selection of natural enemy species for release (Hoelmer and Kirk, 2005), and are a particular concern in California. Olives are grown both in coastal counties, which are characterized by mild temperatures year-round, and in the Central Valley, which has very hot summers and colder winters. The olive Xy thrives in both regions, but we cannot assume

K.R. Sime et al. / Biological Control 39 (2006) 248–255 100 Culture A, female Culture A, male Culture B, female Culture B, male

100 80

a

a Longevity (in days)

Adult longevity (in days)

120

253

60 40

80 60 b

40 20

20

c

c

water

nothing

0

0 15

20

25

30

water honey olives

35

Temperature (°C)

Fig. 3. Adult P. concolor longevity declined with increasing constant temperature for each gender and culture (Culture A, female: y D 51.31–1.231x, r2 D 0.211, df D 1,58, F D 16.84, P < 0.001; Culture A, male: y D 68.10– 1.814x, r2 D 0.439, df D 1,58, F D 47.20, P < 0.001; Culture B, female: y D 154.1–4.488x, r2 D 0.553, df D 1,58, F D 16.84, P < 0.001; Culture B, male: y D 110.6–3.243x, r2 D 0.632, df D 1,58, F D 73.93, I < 0.001). Paired comparisons at each temperature tested show signiWcant diVerences between cultures for female parasitoids at 15 °C (T D 1.557, df D 18, P < 0.001), 22 °C (T D 3.535, df D 18, P D 0.002), 25 °C (T D 3.469, df D 18, P D 0.003), and 32 °C (T D 2.129, df D 18, p D 0.047) and male wasps at 22 °C (T D 2.078, df D 18, P D 0.052), 25 °C (T D 4.324, df D 18, P < 0.001), 28 °C (T D 2.231, df D 18, P D 0.038), and 30 °C (T D 1.849, df D 18, P D 0.081) Within each culture, there were no signiWcant diVerences between female and male longevity at any temperatures tested.

that any single parasitoid species will. The European experience indicates that olive Xy thrives where many parasitoid species do not (Clausen, 1978; Greathead, 1976; Wharton, 1989). Other biological control programs in California have documented the ability of parasitoid species to provide control inland but not on the coast, or vice versa, with the diVerences attributed at least in part to climate (Dahlsten et al., 2005; Yu et al., 1990). Psyttalia concolor from Culture B, with greater longevity at both high and low extremes, appears from this result to be particularly promising for widespread control of the olive Xy in California. 3.3. Adult longevity given diVerent provisions Female P. concolor from both cultures lived longest when provisioned with uninfested olives, honey, and water, or just honey and water (Fig. 4 and Table 2). Provision with water alone, or with nothing, signiWcantly decreased longevity. Life span also declined signiWcantly when hosts were available, suggesting that energy was expended in searching and probing for hosts and producing eggs (Quicke, 1997). 3.4. Lifetime fecundity Average lifetime production of progeny for Culture A was 28.7 § 4.1 adult oVspring obtained per female, which did not diVer signiWcantly from that of culture B (22.2 § 5.1 per female). Progeny production rates were highest during the Wrst 12–14 d and declined thereafter (Fig. 5A), although the parasitoids continued to live to an average of 33.2 § 7.9

water honey

water honey hosts

Fig. 4. Adult P. concolor female longevity was signiWcantly aVected by provisioning (F D 26.35, df D 4, 95, P < 0.001). Treatment means with diVerent letters are signiWcantly diVerent (Tukey’s multiple comparison test, P < 0.05).

Table 2 For each provision treatment tested, there was no signiWcant diVerence between Psyttalia concolor cultures A and B in female longevity (days) Provisions

Culture Mean (§ SEM)

Water, honey, and olives A B Water, honey A B Water, honey, and hosts A B Water A B Nothing A B

65.3 § 8.9 81.3 § 12.0 77.6 § 15.3 68.8 § 16.4 33.2 § 7.9 29.9 § 4.7 8.6 § 1.2 11.6 § 1.1 7.3 § 0.8 9.6 § 1.4

t-statistic P-value 1.071

0.298

0.392

0.699

0.359

0.724

1.889

0.075

1.413

0.174

Two-sample t-test of Culture A and B for each provision treatment, df D 18 for each.

and 29.9 § 4.7 days for Culture A and B, respectively (Figs. 4 and 5B). The mean proportions of female oVspring obtained were 0.64 § 0.07 (A) and 0.59 § 0.08 (B), which do not diVer signiWcantly from each other. The absence of diVerences between the two cultures in total fecundity, in lifetime fecundity patterns, and in the sex ratios of oVspring indicates that they do not diVer in their ability to reproduce on the olive Xy (at least in the laboratory). This result supports those of the previous section, in which it was determined that the two cultures have similar patterns of preference and reproductive success with respect to host stage. Given that both cultures have a similar history, originating with wild stock on olive Xy but reared for many generations on MedXy before being returned for several generations to olive Xy, using similar rearing methodologies throughout, this result is not surprising. The results are similar to those reported for P. concolor on MedXy, where 24–42 oVspring per female may be obtained under varying environmental conditions (Stavraki-Paulopoulou, 1966). They are also not signiWcantly diVerent from results obtained for two Diachasmimorpha species reared under conditions identical to those described

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Offspring per female

A 10 8 6

Culture A Culture B

4 2 0 0

10

20

30

40

50

0

10

20

30

40

50

Number adults alive

B 12 10 8 6 4 2 0

Time (in days)

Fig. 5. (A) Mean lifetime production of oVspring (§SEM) produced by P. concolor when provisioned with hosts, honey and water. (B) The number of adult P. concolor alive at the beginning of each 2-day interval of the trial.

in this study (23.6 § 5.3 and 22.7 § 5.5 for D. longicaudata and D. kraussii, respectively) (Sime et al., 2006). 3.5. Implications for classical biological control In documenting diVerences between two laboratory cultures in longevity under various conditions, this study indicates that variability in such traits must be taken into consideration in selecting and rearing P. concolor for Weld release. The circumstances that caused the observed variation are unknown, but such variation is often attributed to selective regimes imposed by diVerent rearing conditions, variation in the wild stock from which the cultures were obtained (although this should not pertain to our P. concolor cultures), microbial infection, or culture inbreeding (Schmidt et al., 2003). Here, the diVerences between the two cultures are most pronounced at lower temperatures. This Wnding suggests that parasitoids from Culture A had been subject to selection for tolerance of the higher temperatures that the stock was exposed to in Kenya. Regardless of cause, that variation in temperature tolerance is observed at all suggests that it occurs in nature as well. Climate matching of Weld populations of P. concolor to release sites has not been emphasized to date, but ought to be considered in future exploration eVorts. Alternatively, if it is culture inbreeding that leads to changes in adult longevity and temperature tolerance, these shifts ought to be monitored in longstanding cultures. Poor performance in P. concolor col-

onies might be improved by more frequent introduction of wild stock. Genetic analyses aimed at resolving the taxonomy of the P. concolor species complex are currently in progress (R. Stouthamer and R. Wharton, personal communication). Various cultures have been included in their study and one gene region has been sequenced (the D2 expansion segment of the 28s rDNA gene). To date, no genetic distinction has been found for the two cultures discussed in this study. There is no reason to assume that genetic markers such as this one will sort out populations that diVer in behavioral and physiological traits of interest—traits that are likely under the control of other and often many genes (Hopper et al., 1993). Should any such markers be found, however, they could provide a useful tool for identifying populations in the Weld that may have desirable traits. Our results have implications for Weld release protocols as well. Although previous authors have recommended release of P. concolor when olive Xy populations are mostly Wrst and second instars (Raspi and Canale, 2000), our results indicate instead that P. concolor will achieve greater reproductive success in commercial olives when third instar hosts are available. Other parasitoids with longer ovipositors, such as Diachasmimorpha species, may prove better suited for release when younger host stages predominate (Sime et al., 2006). Finally, this study provides information that will be useful in comparing P. concolor with other imported parasitoid species and other populations of P. concolor as potential biological control agents for olive Xy in California. We conWrmed that P. concolor can be successfully reared on olive Xy in olive fruit, and we prefer this culture technique because it is less likely to select for traits that are incompatible with Weld success (Kimani-Njogu et al., 2001). The ease of rearing P. concolor suggests that it will compare favorably to other parasitoids. It should be noted, however, that any such advantage may be negated by the potential of P. concolor to attack non-target host species: there is evidence that P. concolor may have a wider host range under laboratory conditions (Clausen, 1978). The host range of P. concolor is uncertain but it is evidently not a specialist on olive Xy alone, and therefore it is necessary to determine the level of risk its release might pose to other tephritids present in California. These may include a variety of native species as well as introduced beneWcials (Headrick and Goeden, 1996; Sobhian, 1993; Turner et al., 1996), though most of these species feed in seed heads or galls, habitats that may not prove attractive to P. concolor. Further investigation of geographically or biologically distinct populations of P. concolor, which may represent host races, might however help identify populations that are relatively specialized on olive fruit Xy or other fruit pests. Acknowledgments We thank J. Andrews, H. Beeson, C. Funk, L. Miljkovic, S. Mortezaei, H. Nadel, M. Orsini, C. Pickett, and M.

K.R. Sime et al. / Biological Control 39 (2006) 248–255

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