Tritrophic consequences arising from a host shift between apple and walnut in an oligophagous herbivore

Biological Control 65 (2013) 330–337

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Tritrophic consequences arising from a host shift between apple and walnut in an oligophagous herbivore Jana Collatz ⇑, Silvia Dorn ETH Zurich, Institute of Agricultural Sciences/Applied Entomology, Schmelzbergstrasse 9/LFO, 8092 Zurich, Switzerland

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Tritrophic effects of an herbivore shift

from apple to walnut have been studied.  The parasitoid Hyssopus pallidus learns odors from either plant during ontogeny.  Adult wasps reared with apple odors respond to walnut odors but not vice versa.  Parasitoid host acceptance is not affected by juglone in the hosts’ diet.  Only hosts fed on high juglone diet slightly impair parasitoid offspring.

a r t i c l e

i n f o

Article history: Received 9 January 2013 Accepted 21 March 2013 Available online 30 March 2013 Keywords: Hyssopus pallidus Cydia pomonella Host shift Biological control Preimaginal learning Juglone

a b s t r a c t Host plant shifts by herbivorous insects may have fitness consequences beyond the herbivore, cascading up to higher trophic levels, finally affecting natural enemies. Multitrophic effects of a host shift from apple to walnut trees in a disjunctive oligophagous herbivore, the codling moth Cydia pomonella, might thus affect its specialist parasitoid Hyssopus pallidus. Potential consequences for the parasitoid wasps might result from alterations in host finding abilities and effects of the host plant’s defense compound juglone on host acceptance and host quality. Bioassays showed that chemical cues from walnut fruits were as attractive to the wasps as were cues from apple fruits, when parasitoids were reared in the presence of the respective volatile cue. Rearing experiments with levels of juglone mimicking low and high natural concentrations in the hosts’ diet revealed that host acceptance of parasitoids was not affected by juglone while juglone mimicking high levels of natural concentrations caused mild impairment of host quality. Our study suggests that the parasitoid is less affected by the host plant’s defense chemistry than the herbivore and that it is therefore probably able to follow a shift of C. pomonella from walnut to apple. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction Herbivorous insects may shift between host plant species to increase food availability (Sarfraz et al., 2009). Such host shifts may occur frequently during both the lifespan of individual herbivores and, on a larger time scale, also within populations. When a host shift occurs, defense chemistry of plants can play a decisive role in either limiting the range of potential new host plants (e.g. Mao et al., 2006; Lampert et al., 2011) or in sublethally affecting ⇑ Corresponding author. Fax: +41 44 6321171. E-mail address: [email protected] (J. Collatz). 1049-9644/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biocontrol.2013.03.011

performance of the herbivore. Fitness costs resulting from tolerance or detoxification of defense compounds (Cresswell et al., 1992) may then select for adaptations in subsequent generations (Guo et al., 2010) and ultimately lead to the formation of distinct host strains, host races and new species (Becerra, 1997). Host shifts however, can also affect the herbivore’s natural enemies (Ode, 2006). For instance, host finding abilities of natural enemies may vary on the different host plant species (Liu and Jiang, 2003; Tamo et al., 2006), and host acceptance may be impaired either by the presence (Kos et al., 2012) or absence (Kos et al., 2012; Hare and Morgan, 2000) of secondary plant metabolites. Furthermore performance of the natural enemy may be affected

J. Collatz, S. Dorn / Biological Control 65 (2013) 330–337

directly by plant defense compounds or indirectly by a reduction of host quality, due to detoxification costs of such compounds (Sarfraz et al., 2009; Moreau et al., 2009). Multitrophic effects of a host shift may be more pronounced the more distantly related, i.e. the more different in their defense chemistry the host plant species are. Therefore systems with disjunctive oligophagous herbivores, which feed on few unrelated plant species that might strongly differ in chemical composition, are interesting subjects to study in this context. One such example is the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae). Its main host plants are apple trees (Hausmann et al., 2005) but host shifts from apple to walnut occur, as has been shown by studies revealing a certain gene flow between codling moth populations of the two host species (Chen and Dorn, 2010). Both apple and walnut trees have likely co-evolved with the codling moth in Central Asia (Juniper and Mabberley, 2006; Grisa et al., 2008). The host shift of the codling moth is presumably facilitated by a sufficient overlap between emitted volatile constituents, including several mono- and sesquiterpenes as well as green leaf volatiles and nonanal, in the two host plants (Casado et al., 2008; Witzgall et al., 2005; Vallat and Dorn, 2005) and promoted by alternation in fruit bearing of apple trees. C. pomonella caterpillars develop in the green husks surrounding the walnut, which are heavily protected by the presence of naphtoquinones. Their main constituent juglone, which is toxic to many insect species (Thiboldeaux et al., 1994; Lindroth et al., 1990), was quantified in walnut husks at concentrations between 2 (Stampar et al., 2006) and 19 mg/g of dry weight (Radix et al., 1998). C. pomonella caterpillars reared on a diet containing juglone at such natural concentrations were able to detoxify and excrete it. However, some fitness traits, i.e. developmental time, weight gain and survival were moderately affected in a dose dependent manner (Piskorski and Dorn, 2011). A natural enemy that could be affected by a host shift of the codling moth is the gregarious larval ectoparasitoid Hyssopus pallidus (Askew) (Hymenoptera: Eulophidae), which is native to Europe (Tschudi-Rein et al., 2004; Askew, 1964). H. pallidus is a candidate agent for biological control of the codling moth (Mattiacci et al., 1999; Dorn, 2005). To find its host, the female wasp enters the infested fruit through the feeding tunnel burrowed by the caterpillar, or in apple also through the calyx, irreversibly paralyzes the host, and deposits several eggs onto its body. H. pallidus is assumed to have evolved in the apple system (Hausmann et al., 2005), however, it possesses remarkable behavioral plasticity relating to chemical cues from the host’s food plants (Gandolfi et al., 2003a). After exposure to host or even non-host plant volatiles during development, the females prolonged their searching time on the same cue in the adult stage significantly (Gandolfi et al., 2003b). Therefore, this parasitoid can likely explore host caterpillars on a wider range of plants. In the present study we investigated how the host shift of the herbivore that displays disjunctive oligophagy between its two phylogenetically distant food plants apple and walnut, affects behavioral and physiological parameters of its ectoparasitoid H. pallidus. First, we tested for the influence of olfactory cues from apple and walnut on the searching behavior of adult female wasps. As plant volatiles, to which the parasitoid is typically exposed during its preimaginal phase in the field, can strongly influence adult host searching in H. pallidus (Gandolfi et al., 2003a), we provided either walnut or apple volatiles during the parasitoid development. Second, we investigated how juglone, the main defense compound of walnut in the host caterpillar’s diet (Piskorski and Dorn, 2011), affects host acceptance by the female wasp. Third, we investigated how the host quality of juglone-fed caterpillars is altered, by assessing key performance parameters of the parasitoid, i.e. developmental time, number of offspring per host as well as sex ratio, body size, lifespan and fecundity of offspring.

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2. Material and methods 2.1. Insect cultures H. pallidus was maintained as a single stock colony under controlled conditions (16:8 h L:D; 22 ± 2 °C and 60% RH). The stock colony, established in 1996, originated from France (Tschudi-Rein and Dorn, 2001) and continued to be regularly genetically supplemented (Häckermann et al., 2007; this study) by the introduction of field-collected adult wasps from apple orchards in Switzerland. The colony was reared on C. pomonella host caterpillars, which in turn were reared on a soy-flour-based artificial diet (Najar-Rodriguez et al., 2013). In the moth rearing, adults were collected on the day of eclosion and transferred to a paper cylinder with moist cotton for oviposition (Hern and Dorn, 1999). Upon hatching, caterpillars were transferred to Petri dishes, allowed to feed on the artificial diet, and then offered corrugated cardboard strips for pupation. For parasitization, 30 fifth-instar caterpillars were offered at a ratio of one host per mated H. pallidus female parasitoid in a plastic box (10  10  8 cm) (Häckermann et al., 2008). The boxes contained two mesh-covered holes to ensure for sufficient ventilation, and honey was offered as a food source for the adult wasps. Since plant olfactory cues present in the preimaginal environment of the parasitoid influence the behavior of the adult wasp, irrespective of whether the cue is offered during the ontogeny of an individual specimen or during several generations (Gandolfi et al., 2003a), small pieces of either fresh apple or green walnut husk were added. These were placed at approximately 4 cm distance from the paralyzed hosts during the entire preimaginal development of the ectoparasitoid. Upon emergence, the apple- or walnut-exposed parasitoids were transferred into separate Plexiglas cages (25  25  25 cm) and maintained with water and honey until use in bioassays. 2.2. Diet supplemented with the walnut defense compound juglone To investigate effects of the targeted secondary walnut metabolite, juglone (purity P97%; Sigma–Aldrich, Buchs, Switzerland) was added at concentrations of 5 and 25 mg per gram dry weight to the diet of C. pomonella, to encompass low and high juglone concentrations in walnut husks, respectively (Radix et al., 1998; Stampar et al., 2006). Juglone was added at the end of diet preparation; the diet was homogenized and distributed into Petri dishes (55 mm diameter) at a 4 mm thick layer. The diet was prepared 24 h before host caterpillars were introduced, and chemical stability of juglone using this method has been documented by Piskorski and Dorn (2011). Juglone-free diet was used as control. Unfed first instar caterpillars (6 ± 6 h-old) were transferred singly with a fine brush into the Petri dishes. 2.3. Response of female wasps to fruit chemical cues of phylogenetically distant hosts 2.3.1. Plant material Fruits without any visible damage were collected from unmanaged walnut and apple trees in late July and early August 2011. Intact walnut fruits (Juglans regia (L.), Juglandaceae; mixed varieties), i.e. still completely covered by their husk, were collected from park trees in Zurich city-area (47°230 N, 8°310 E, elevation 475 m). Intact apple fruits (Malus domestica (L.), Rosaceae, mixed varieties) were collected from an untreated organic orchard in northern Switzerland (Kirchdorf: 47°300 N, 8°170 E, elevation 552 m). Fruits were either used for infestations in the laboratory with neonate codling moth caterpillars in order to collect their feces subsequently for

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extraction, or directly for extraction to obtain fruit extracts within 24 h of collection. Previous studies had shown that the parasitoid H. pallidus searches for a host in the fruit particularly on areas contaminated with host frass (Mattiacci et al., 1999), and that an extract of such frass elicits a similar behavior (Gandolfi et al., 2003b). 2.3.2. Preparation of fruit and frass extracts and bioassays To examine to which degree chemical cues from walnut can be used during host location by the parasitoid, single choice-bioassays, based, on the procedure developed by Gandolfi et al. (2003b), were performed on fruit and host frass extracts, which were prepared from apple fruits or walnut fruits, respectively. Fruit material was cut into small pieces of 0.2–0.3 mm (comprising the fruit constituents with which the caterpillars are usually in contact; for apple: 9% skin, 85% pulp and 6% seed material (Gandolfi et al., 2003b); for walnut: 9% skin, 85% husk pulp and 6% nutshell). The material from each plant was extracted in diethyl ether (purity P99.8%) at 0.2 g fruit material per ml for 18 h. Both extracts were concentrated tenfold in a warm water bath (50 °C) before use in bioassays. Frass material expelled from caterpillar-infested apple fruit (termed apple frass) or caterpillar-infested walnut fruit (termed walnut frass) was collected and air-dried for 48 h. Apple frass and walnut frass were extracted at 0.13 g/ml diethyl ether for 18 h (Gandolfi et al., 2003b). All extracts were filtered through pre-cleaned cotton wool before use in bioassays. In the field, host searching adult females are always exposed to frass from fruit-feeding caterpillars. Accordingly, we treated filter papers with frass extract from fruit-fed caterpillars and presented them to the female wasps. Filter paper halves (3 cm diameter, no. 595, Schleicher & Schuell, Dassel, Germany) were placed on a glass plate and treated with 20 ll extract. The solvent was allowed to evaporate for 3 min. The filter papers were then transferred to a glass Petri dish (5 cm diameter). Since previous trials have shown that searching time of adult parasitoid females did not significantly differ irrespective of whether exposed to frass from apple-fed caterpillars or to apple cues alone (Gandolfi et al., 2003b), we simplified adult bioassays, after confirmation of this basic relationship in both of our systems, by offering the fruit extracts only. Behavioral bioassays were conducted between 4 h and 10 h after onset of photophase at 22 ± 1 °C and 50 ± 5% RH with 4–7-day-old mated parasitoid females, which had experienced apple or walnut volatile chemical cues during their preimaginal development. Immediately prior to the bioassay parasitoids were transferred individually from the emergence cages into plastic vials (3 cm diameter; 1.5 cm height). The lid of the plastic vial (3 cm diameter; 0.5 cm height) with the female sitting on its inner surface was then gently moved to finally cover the filter paper on the glass Petri dish. The observation started once the parasitoid began to move and ended 600 s later. Using the software ‘‘The Observer XT 8.0’’, Noldus Information Technologies (2008) the behavior of the wasp (searching: defined as walking with intensive antennal examination of substrate; resting) as well as its location (on filter paper; other) were recorded. The accumulated searching time on the filter paper was taken as measure for arresting properties of the odor source (Gandolfi et al., 2003b). We quantified the responses of wasps reared in the presence of volatiles from apple and walnut fruits to extracts from fruit-fed caterpillars, or from fruits, as well as to a solvent control. Wasps that did not move for more than half of the total observation time (300 s) were considered non-responsive and were excluded from the analysis. 2.4. Effect of the secondary metabolite juglone on H. pallidus host acceptance To investigate to which degree the walnut defense compound juglone in the diet of the host caterpillar affects host acceptance

by H. pallidus, adult parasitoids were given access to host caterpillars reared on diet containing different levels of juglone. To obtain last instar caterpillars suitable for parasitism, we reared caterpillars individually applying three different treatments: reared on diet devoid of juglone (J0 caterpillars) or supplemented with either 5 or 25 mg per gram dry weight (J5 or J25 caterpillars, respectively). To obtain last instar caterpillars at the same time irrespective of the juglone concentration in the diet, we compensated for slower development on the high juglone level (Piskorski and Dorn, 2011) by setting up J25 caterpillars 3–5 days ahead of J5 and J0 caterpillars. Only last (fifth) instar caterpillars (Blomefield and Giliomee, 2009) with a similar body mass across the three treatments were used (mean ± s.e.: J0 caterpillars 42.7 ± 1.0 mg; J5 caterpillars 42.6 ± 1.1 mg; J25 caterpillars 42.3 ± 1.0 mg; no significant difference between the three treatments Kruskal–Wallis: H2 = 0.2; p = 0.892). They were transferred singly into transparent plastic vials (35 mm diameter, 80 mm height), which contained corrugated cardboard, honey and a small piece of fruit (to stimulate parasitism), as well as a hole covered with plastic gauze to allow air ventilation. Experiments with parasitoids were conducted at 16:8 h L:D; 25 ± 2 °C and 60% RH. To allow for mating and subsequent parasitism a randomly chosen 4-day-old male and female wasp were introduced into the vials. We investigated the potential impact of the three juglone treatments (105 vials each) on the time period until host acceptance by evaluating host acceptance at 24 h intervals for seven days following parasitoid introduction. At each daily assessment time, a batch of 15 vials was removed and evaluated. The number of parasitoid eggs and/or larvae on the host caterpillar was counted under a dissecting microscope and the proportion of parasitized caterpillars and the mean number of eggs and larvae per parasitized caterpillar was taken as measure for host acceptance. 2.5. Effect of juglone on parasitoid development and offspring quality To assess effects of juglone on selected key performance parameters of H. pallidus, parasitoids were reared on caterpillars fed with the juglone-free or the two juglone-supplemented diets. Rearing of caterpillars and setup of parasitoid wasps were conducted as described in the experiment on host acceptance, but all wasps were removed from the experimental vials 24 h after setup (n = 185). Again, only caterpillars with a similar body mass across the three treatments were used. By chance, the mean body mass attained slightly higher values than in the preceding experiment (mean ± s.e.: J0 caterpillars 60.97 ± 1.1 mg; J5 caterpillars 60.92 ± 1.2 mg; J25 caterpillars 60.04 ± 1.2 mg; no significant difference between the three treatments Kruskal–Wallis: H2 = 0.4; p = 0.831). Vials were observed at 24 h intervals for the emergence of parasitoids, and parasitoid developmental time was assessed as the number of days from introduction of parent wasps into the vial until offspring emergence. Number and sex of wasps emerging per parasitized host were recorded. The proportion of females per clutch (i.e. the secondary sex ratio of females to males) was assessed. As a proxy of body size, we assessed the mean hind tibia length of five females per clutch at 50 magnification under a Wild M3B stereomicroscope (former Wild Heerbrugg AG, Switzerland) equipped with an ocular micrometer with a scale etched with divisions of 0.016 mm (n = 45 per treatment). To quantify lifespan, mated female progeny from the three treatments were kept in plastic vials (45 mm diameter, 100 mm height), supplemented with a droplet of honey and a moist strip of filter paper, without access to hosts. Filter paper and honey were exchanged regularly. The number of surviving wasps was assessed at 48 h intervals until all wasps had died (n = 31).

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3. Results 3.1. Response of female wasps to fruit chemical cues of phylogenetically distant hosts

R

25

Searching time [s]

200

3.2. Effect of the secondary metabolite juglone on H. pallidus host acceptance Host acceptance was not influenced by natural concentrations of juglone (J5, J25) in the diet the host caterpillars had fed on. Over-

b

a

150 100 50 0

B

24

b

Dev. on walnut

250

Control

Apple frass

Apple

22

27

21

R

b

b

200 150

a

100 50 0

Control

C

250 200

R

26

28

150

b

100

Walnut

Walnut frass

a

50 0

D

250 200

R 24

21

a a

150 100 50 0

Walnut

Both extracts from frass of apple-fed caterpillars (termed apple frass) and from apple fruits elicited significantly prolonged searching activity of parasitoids that had developed in the presence of apple odor compared to the control without apple fruit cues (Fig. 1A; H2 = 15.1, p = 0.001; apple extract: U = 15.5, p = 0.013; apple frass: U = 23.4, p < 0.001). The use of apple fruit extract led to the same increase in response as the use of apple frass extract (U = 7.9, p = 0.599). Likewise, both extracts from frass of walnut-fed caterpillars (termed walnut frass) and from walnut fruits elicited significantly prolonged searching activity of parasitoids that had developed in the presence of walnut odor compared to the control without walnut fruit cues (Fig. 1B; H2 = 19.6, p < 0.001; walnut extract: U = 20.2, p = 0.001; walnut frass: U = 24.9, p < 0.001). The use of walnut fruit extract led to the same increase in response as the use of walnut frass extract (U = 4.7, p = 0.430). Wasps that had developed in the presence of apple odor and that were tested as adults on walnut odor displayed a searching time almost twice as long (179%) as the searching time of parasitoids exposed to the opposite combination of cues, i.e. wasps that had developed in the presence of walnut odor and that were tested as adults on apple odor (Fig. 1C; U = 545.5; p = 0.002). In contrast, searching time was not significantly different on cues of the two fruits if the adult wasp was searching on the same fruit cues it had been exposed to during its preimaginal development, although searching time on walnut cues was longer than on apple cues (Fig. 1D; U = 173.0; p = 0.072).

26

Searching time [s]

For all data sets normality of data was checked using Shapiro test and equality of error variances using Levene’s test of equality of error variances. Due to heteroscedasticity in the data sets multiple comparisons were done by non-parametric Kruskal–Wallis tests followed by Mann–Whitney post hoc tests with false discovery rate correction. Single comparisons were done by Mann–Whitney test. All statistical analyses were performed using the software SPSS Statistics 19 (IBM SPSS Statistics, Chicago, IL, USA, 2010).

250

Dev. on apple

Searching time [s]

2.6. Statistical analysis

A

Searching time [s]

To assess fecundity of the progeny, 4-day-old male and female wasps from each of the three treatments were paired and given access to juglone-free reared fifth instar caterpillars as described above (n = 31). Caterpillars were weighed before the introduction of parasitoids, and assignment of individual wasps to the caterpillars was randomized. The paired male and female wasps always originated from a different clutch, and only one or two female wasps from the same clutch were used in the experiment. Every four days the female wasp was transferred to a fresh host, and this procedure was repeated until the wasp died. Previous trials had shown that egg maturation in this synovigenic species is a slow process, with females requiring up to 3 days to produce a new set of mature eggs (Tschudi-Rein and Dorn, 2001). We focused on the offspring number on the first four parasitized caterpillars, as preliminary trials had shown that the bulk of wasp offspring was deposited on the first four hosts.

Apple

Apple

Walnut

Fig. 1. Response of female Hyssopus pallidus wasps to solvent extracts of frass of apple- or walnut-fed Cydia pomonella caterpillars, or to extracts of apple or walnut fruits. Bars indicate searching time (mean ± s.e.) on each treatment in single-choice bioassays. (A) Parasitoids that had developed in the presence of apple odor (Dev. on apple) tested as adults on solvent control (Ad control), apple-fed C. pomonella frass extract (Ad apple frass) and apple extract (Ad apple); (B) parasitoids that had developed in the presence of walnut odor (Dev. on walnut) tested as adults on solvent control, walnut fed C. pomonella frass extract (Ad walnut frass) and walnut extract (Ad walnut); (C) parasitoids that had developed in the presence of apple odor tested as adults on walnut extract; and parasitoids that had developed in the presence of walnut odor tested as adults on apple extract; (D) parasitoids that had developed in the presence of walnut odor tested on apple extract; and parasitoids that had developed in the presence of walnut odor tested on walnut extract. N = 30 per treatment. Multiple comparisons were analyzed by Kruskall–Wallis-tests followed by Mann–Whitney post hoc tests with false discovery rate correction; single comparisons were analyzed by Mann–Whitney tests. Different letters indicate significant differences between treatments. R: Number of responding individuals.

all, neither the proportion of host caterpillars parasitized, nor the number of eggs deposited per parasitized caterpillar differed significantly from the control, where the caterpillars had fed on juglone-free diet (J0) (proportion of host caterpillars parasitized: H2 = 0.9; p = 0.641; eggs deposited per parasitized caterpillar H2 = 0.03; p = 0.983). Likewise, values were not significantly different at any of the investigated durations of host access (1–7 days, Table 1). Although the trial design allowed parasitoids exposed to a host for four days and more to deposit a second batch of eggs, this potential self-superparasitism, which was detectable by offspring batches of two distinct body sizes, did not result in significantly increased number of eggs per parasitized caterpillar (e.g. total number of eggs on day 3 vs. total number of eggs on day 7: overall: U = 539.5, p = 0.653).

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Table 1 Host acceptance of Hyssopus pallidus parasitoids on Cydia pomonella caterpillars, which had fed on diet devoid of juglone (J0), on diet supplemented with 5 mg/g dw juglone (J5), or on diet supplemented with 25 mg/g dw juglone (J25). Duration of host access [d] 1

2

3

4

5

6

7

Proportion of hosts parasitized

J0 J5 J25

0.33 0.33 0.33

0.40 0.53 0.40

0.67 0.67 0.73

0.67 0.81 0.75

0.73 0.80 0.80

0.80 0.87 0.87

0.80 0.80 0.80

Number of eggs per parasitized host (mean ± s.e.)

J0 J5 J25

9.8 ± 1.5 11.6 ± 2.1 10.4 ± 2.2

11.7 ± 2.8 9.0 ± 1.8 12.2 ± 1.6

12.9 ± 1.8 14.5 ± 1.0 13.4 ± 1.7

15.2 ± 1.9 12.5 ± 1.8 13.8 ± 1.5

14.1 ± 1.3 16.2 ± 1.7 14.9 ± 1.4

13.1 ± 2.1 14.6 ± 1.7 12.3 ± 1.0

13.4 ± 1.7 12.8 ± 1.8 12.9 ± 1.9

Sample size: 15 per treatment; Kuskall–Wallis-tests: no significant differences between treatments (proportion of parasitized hosts: overall: H2 = 0.9; p = 0.641; eggs per parasitized host: overall: H2 = 0.03; p = 0.983; day 1: H2 = 0.3; p = 0.854; day 2: H2 = 1.7; p = 0.421; day 3: H2 = 1.9; p = 0.392; day 4: H2 = 2.1; p = 0.357; day 5: H2 = 0.8; p = 0.676; day 6: H2 = 0.3; p = 0.880; day 7: H2 = 0.4; p = 0.818).

3.3. Effect of juglone on parasitoid development and offspring quality Developmental time of parasitoids was significantly prolonged by about 0.7 d on J25 caterpillars (Fig. 2A; H2 = 39.5, p < 0.001) compared to J0 caterpillars (U = 60.9, p < 0.001) and J5 caterpillars (U = 56.3, p < 0.001). Number of offspring produced per parasitized caterpillar was not significantly affected by the presence or level of juglone in the caterpillars’ diet (Fig. 2B; H2 = 1.6, p = 0.454). Visual evaluation of moth carcasses after emergence of wasps revealed the presence of dead immature stages of parasitoids in only one of 195 cases.

B

16 b

15.5 15 a

a

14.5

Offspring per host

Development time [d]

A

14

D

1 0.95 a

a

a

0.9 0.85

60 a

a

50

Total fecundity

F

70 b

Lifespan [d]

a

a

14

4. Discussion

12

0.5

0.46

a

a

b

0.42

0.38

0.8

E

a 16

10

Tibia length [mm]

Females : males

C

18

90

70

a

a b

50

30

40 Control

Juglone Juglone 5 mg/g dw 25 mg/g dw

The proportion of females (i.e. the ratio of females: males) per batch of offspring was not significantly affected by the presence or level of juglone in the host’s diet (Fig. 2C; H2 = 0.3, p = 0.864). The mean tibia length of female offspring was significantly shorter (9%) in parasitoids that had developed on J25 caterpillars (Fig. 2D; H2 = 39.6, p < 0.001) compared to those that had developed on J0 caterpillars (U = 348.5, p < 0.001) and on J5 caterpillars (U = 1686.0, p < 0.001). Adult female lifespan of F1 offspring that had developed on J5 caterpillars was significantly prolonged by 5 or 10 days, respectively (Fig. 2E; H2 = 7.1, p = 0.029) compared to offspring that had developed on J0 caterpillars (U = 621.0, p = 0.048) and J25 caterpillars (U = 662.5, p = 0.010). Fecundity of parasitoid offspring was significantly reduced by about one quarter in parasitoids that had developed on J25 caterpillars (Fig. 2F; H2 = 8.986, p < 0.011) compared to J0 (U = 16.9, p < 0.013) and J5 caterpillars (U = 18.5, p = 0.021).

Control

Juglone Juglone 5 mg/g dw 25 mg/g dw

Fig. 2. Development and offspring quality of Hyssopus pallidus parasitoids that had developed on Cydia pomonella larvae fed juglone-supplemented or juglone-free diet. (A) Development time (mean ± s.e.) on hosts from each treatment (n = 78, 78, 78); (B) number of offspring per parasitized host (mean ± s.e.) from each treatment (n = 112, 110, 99); (C) female to male ratio (mean ± s.e.) on hosts from each treatment (n = 111, 107, 93); (D) female tibia length (mean ± s.e.) of parasitoid offspring from hosts from each treatment (n = 45, 45, 45); (E) lifespan (mean ± s.e.) of parasitoid offspring from hosts from each treatment (n = 31, 31, 31); (F) total fecundity (mean ± s.e.) of parasitoid offspring from hosts from each treatment on the first four hosts parasitized (n = 31, 31, 31). Different letters indicate significant differences between treatments (Kruskall–Wallis-tests followed by Mann–Whitney-U post hoc tests with false discovery rate correction).

Both the parasitoid’s behavior and its physiological parameters could be affected when its oligophagous herbivore host shifts from the primary to a phylogenetically distant secondary food plant. Using the system composed of the plants apple and walnut, C. pomonella caterpillar hosts and the ectoparasitoid H. pallidus, we showed that wasp’s behavior was not affected, as females responded positively to volatiles of the secondary food plant as well, and they accepted the host caterpillars regardless of presence or absence of the walnut secondary metabolite juglone in their diet. However, parasitoid offspring was mildly affected by a high level of juglone in the host’s diet. 4.1. Response of female wasps to fruit chemical cues of phylogenetically distant hosts Fruit cues are important for promoting host finding in the ectoparasitoid H. pallidus (Gandolfi et al., 2003b). We confirmed the findings by Gandolfi et al. (2003b) showing that preimaginal exposure of the parasitoid to apple volatiles enhanced the searching activity of adult females on chemical cues from apple frass and apple, and we newly found an analogous response to chemical cues from walnut frass and walnut after preimaginal exposure of the parasitoid to walnut volatiles. Thus, our findings indicate that this parasitoid possesses the necessary preadaptations for true preimaginal learning of odors from the phylogenetically distant plant walnut. In contrast, selection over generations was necessary for the utilization of host finding cues from a plant outside the range of the host caterpillars’ typical food plant family in the parasitoid Diadegma semiclausum (Hellen) (Rossbach et al., 2006).

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Interestingly, searching time on a foreign cue was clearly more favorable when parasitoids had developed on apple and were presented walnut in the bioassay than vice versa. This finding indicates that the volatiles from apple and walnut are learned differentially during development, although the volatile profiles from both plant species overlap (Casado et al., 2008; Witzgall et al., 2005). In the apple blossom weevil, Anthonomus pomorum (L.), a blend of six volatile compounds released from apple twigs sufficed for weevil attraction, thus the further plant-emitted compounds might be redundant (Collatz and Dorn, 2013). Similarly we assume that H. pallidus uses a distinct combination of few essential compounds from the odors experienced and learned during ontogeny. If this combination includes certain compounds exclusively emitted by walnut, adult parasitoids would likely show a limited recognition of apple volatiles. Conversely, if this combination comprised exclusively volatile compounds from apple, which are shared by walnut, then females would show a similarly enhanced searching response on either apple or walnut. Such differential learning pattern of apple and walnut cues might underlie our results. 4.2. Impact of the secondary metabolite juglone on host and parasitoid C. pomonella caterpillars are directly exposed to the toxic juglone while feeding on walnut fruits. However, they are able to detoxify this secondary metabolite in their intestinal system, and thus are able to establish populations on walnut (Piskorski and Dorn, 2011). Since detoxification efficiently prevents the uptake of juglone into the hemolymph of caterpillars at levels detectable by gas chromatography–mass spectrometry (Piskorski and Dorn, 2011), parasitoids of C. pomonella are not directly exposed to juglone in their food source. In turn, they experience a host, of which the nutritional quality might be altered (Vinson and Barbosa, 1987) due to the costs involved in detoxification. Such costs become apparent in the caterpillar as prolonged developmental time, reduced adult weight and distorted sex ratio (Piskorski and Dorn, 2011). We assume that effects of a reduced host quality on the parasitoid are likely to be less severe than direct toxic effects, or even combined effects of toxins and reduced host quality. Several studies have explored the effects of plant secondary metabolites on tritrophic systems, with various outcomes, both for the parasitoid and the herbivore. Often the relationship between direct and indirect effects of plant secondary metabolites on the higher trophic level could not be as clearly disentangled as in our study. A weaker effect of plant defense compounds on the parasitoid than on its host was demonstrated in a study with Microplitis mediator (Haliday) developing on Mamestra brassicae (L.). This result was attributed to the relatively short duration of intense host-parasitoid relationship, which is terminated with the parasitoid’s eclosion at an early host stage (Harvey and Gols, 2011). In another study, feeding low doses of gossypol to Heliothis virescens (Fabricius) caterpillars resulted in stimulated growth of the herbivore as well as of its parasitoid Campoletis sonorensis (Cameron). High doses of gossypol, however, prevented complete development of H. virescens, while this caterpillar was still suitable as a host for the parasitoid, although its weight was reduced and its developmental time prolonged (Gunasena et al., 1989). Also the opposite has been observed: Nicotine reduced survival of the parasitoid Cotesia congregata (Say), while its host Manduca sexta (L.) was hardly affected (Barbosa et al., 1991). The parasitoid Hyposoter exiguae (Viereck) could be poisoned by a-tomatin acquired from its less sensitive host Heliothis zea (Boddie) (Campbell and Duffey, 1979). Xanthotoxin reduced clutch size of Copidosoma sosares (Walker) without any observable effects on the quality of its host Depressaria pastinacella (Duponchel) (Lampert et al., 2008, 2011).

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In contrast to those other parasitoid species investigated, H. pallidus is an idiobiont parasitoid, which arrests development of its host and just consumes the host at the physiological state reached at the time of parasitization. This might contribute to the finding that H. pallidus was less affected by the plant secondary metabolite than the koinobiont parasitoids mentioned in the previous paragraph. Those parasitoids had to deal with altered developmental pattern or continuous altered nutrient turnover in their host that was confronted with plant defense compounds. 4.3. Effect of the secondary metabolite juglone on H. pallidus host acceptance Parasitoids can assess the internal chemistry of their hosts by ovipositor probing and thus decide whether to accept or reject a host (Powell et al., 1998; Hatano et al., 2008). H. pallidus has to inject the paralyzing venom into the host caterpillar prior to egg deposition onto its body (Dorn and Beckage, 2007), hence an early direct ovipositor contact with host hemolymph is given in this species. However, juglone at natural levels in the caterpillar’s diet did not compromise host acceptance by the parasitoid, although H. pallidus is able to accurately measure the nutritional quality of an encountered host and adjusts the clutch size accordingly (Häckermann et al., 2008). We assume that the indirect effects of juglone posed on the parasitoid, which are based on reduced host quality, are not strong enough to affect host acceptance. 4.4. Effect of juglone on parasitoid development and offspring quality Our results show that developmental time of parasitoids was prolonged when reared on caterpillars that were fed a concentration of juglone mimicking high levels of natural concentrations in walnuts. Despite the ecdysoid inhibiting properties of juglone (Mitchell and Smith, 1988) prolonged developmental time can probably be attributed to a compensation for lower food quality (e.g. Bezemer and Mills, 2001). Mortality of immature stages of parasitoids was negligible irrespective of the diet the hosts were fed with. Offspring number of H. pallidus, which therefore also reflects clutch size, was not significantly reduced by juglone in the host’s diet. It is known from many parasitoid wasps that they adjust the sex ratio of their offspring according to the quality of their host (e.g. Werren, 1983). In some parasitoids also the hosts’ food plants have an influence on the sex ratio (Hare and Luck, 1991; Bhatt and Singh, 1991), which, in the case of Epidinocarsis diversicornis (Howard), may even lead to competitive displacement of the species by the closely related Epidinocarsis lopezi (De Santis) (Gutierrez et al., 1993). However, no difference in the wasp’s secondary sex ratio between offspring from caterpillars fed juglone-free or juglone-containing diet was observed in H. pallidus. Here, secondary sex ratio coincided with primary sex ratio, since preimaginal mortality was negligible. However, a high juglone level in the host’s diet resulted in decreased offspring quality, apparent as diminished body size and reduced fecundity. Thus, the decreased host quality predominantly translates into effects on the second generation of wasps. Other studies on H. pallidus measured fecundity of the parasitoid by multiplying the average number of eggs a female laid per clutch with the total number of hosts attacked by the most successful female (Zaviezo and Mills, 1999; Häckermann et al., 2007). In the present study we opted for the total number of offspring on the first four parasitized hosts as a measure for fecundity, since this corresponds to the mean number of hosts parasitized during a female’s lifespan, and it represents the bulk of offspring produced. As it is unlikely that females will realize all their reproductive potential in nature and as being killed by the host also poses a risk under natural cir-

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cumstances (Mattiacci et al., 2000) this focus seems to be adequate. Whereas the current study found an effect of the critical secondary metabolite at high concentration on body size but not on secondary sex ratio of parasitoid offspring, another study focusing on a potentially toxic primary plant metabolite reported no effect on either offspring parameter in a system composed of dry bean, the bruchid Acanthoscelides obtectus and its pteromalid parasitoid Dinarmus basalis (Velten et al., 2007). Remarkably, in both the current and the previous biological control systems the potentially toxic plants constituent did not penetrate from the digestive system into the hemolymph at measurable rates (Piskorski and Dorn, 2011; Paes et al., 2000). The low juglone level in the host’s diet did not exhibit any significant negative effects on the parasitoid, while the high juglone level affected several fitness parameters. Furthermore, it could be shown that the lifespan of wasps derived from hosts fed the low juglone level diet was even prolonged. It seems that up to a certain juglone threshold either host quality is not affected or the parasitoid is able to compensate for the impaired host quality. The first option is supported by the fact that juglone also possesses antimicrobial and antifungal activities (Yakubovskaya et al., 2009) and could therefore even improve the quality of the host (see also Piskorski et al., 2011). There are also some indications in favor of the second option, as costs of detoxification of juglone in the caterpillar could render the host less capable of defending itself against parasitoid attack (Benrey and Denno, 1997; Smilanich et al., 2009). These beneficial effects could counterbalance effects of reduced quality. 4.5. Conclusion The moderate effects exhibited by the secondary metabolite juglone from the first trophic level on the host caterpillar at the second trophic level did not cascade up to the third trophic level regarding parasitoid host acceptance, offspring number and survival, as well as offspring sex ratio, and they nearly faded off considering parasitoid development time. Hence, the potential use of H. pallidus as a biocontrol agent of C. pomonella in apple might find a promising expansion in the walnut system. Provided the parasitoids are given a preimaginal exposure to walnut olfactory cues prior to release, they would be well conditioned to contribute to a more sustainable walnut cultivation. Acknowledgments We thank Dr. Claudio Sedivy, Dr. John Hoffmann and two anonymous reviewers for helpful comments to the manuscript. References Askew, R.R., 1964. On the biology and taxonomy of some European species of the genus Elachertus Spinola (Hymenoptera, Eulophidae). Bull. Entomol. Res. 55, 53–58. Barbosa, P., Gross, P., Kemper, J., 1991. Influence of plant allelochemicals on the tobacco hornworm and its parasitoid, Cotesia congregata. Ecology 72, 1567– 1575. Bhatt, N., Singh, R., 1991. Bionomics of an Aphidiid parasitoid Trioxys indicus Subba Rao and Sharma (Hym, Aphidiidae). 32. Effect of food plants on the functionalresponse, area of discovery and sex-ratio of F1 offspring at varying densities of the host Aphis gossypii Glover (Hem, Aphididae). J. Appl. Entomol. 111, 263–269. Becerra, J.X., 1997. Insects on plants: macroevolutionary chemical trends in host use. Science 276, 253–256. Benrey, B., Denno, R.F., 1997. The slow-growth-high-mortality hypothesis: a test using the cabbage butterfly. Ecology 78, 987–999. Bezemer, T.M., Mills, N.J., 2001. Walnut development affects chemical composition and codling moth performance. Agric. Forest Entomol. 3, 191–199. Blomefield, T.L., Giliomee, J.H., 2009. Head capsule widths and the rate of development of the instars of codling moth, Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae). Afr. Entomol. 17, 28–33.

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