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Does parasitoid state affect host range expression?
Biological Control 78 (2014) 15–22
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Does parasitoid state affect host range expression? W.H. Jenner a, U. Kuhlmann a, J.H. Miall b, N. Cappuccino c,⇑, P.G. Mason b a b c
CABI Europe – Switzerland, 1 Rue des Grillons, Delémont CH-2800, Switzerland Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada
h i g h l i g h t s 3-d-old & 10-d-old Diadromus
pulchellus were offered 12 non-targets in no-choice & choice trials. 3-d-old & 10-d-old parasitoids attacked four non-target species & offspring emerged from three. Increasing non-target density altered attack by 10-day-old, but not 3-dayold, parasitoids. Parasitoid state influenced frequency of non-target attack but did not affect species attacked.
a r t i c l e
i n f o
Article history: Received 4 March 2014 Accepted 14 July 2014 Available online 24 July 2014 Keywords: Acrolepiopsis assectella Classical biological control Diadromus pulchellus Experimental design Host specificity Non-target Physiological state
g r a p h i c a l a b s t r a c t Pupal parasitoid Diadromus pulchellus on leek moth hosts and non-target species or
or 3-d-old
10-d-old
14 non-target host species in separate no-choice tests
• No effect of parasitoid age on acceptance or mortality of non-target hosts.
3-d-old
One host pupa plus one nontarget pupa in choice test
10-d-old
or
One host pupa plus four nontarget pupae in choice test
• Aack rates by 3-d-old wasps did not depend on non-target density. • 10-d-old wasps were more likely to accept the non-target at higher densies.
a b s t r a c t The pre-release risk assessment of parasitoids for classical biological control generally involves nontarget testing to define the agent’s host range. To ensure that no suitable host species are falsely rejected in these tests, it has been suggested that the physiological and informational state of parasitoids be manipulated to enhance their ‘‘motivation to oviposit’’. However, the effects of such factors on host acceptance are not consistent across parasitoid species, making it laborious to identify the conditions necessary to maximise host acceptance. Our objective was to determine whether changes in parasitoid state could alter host acceptance behaviour sufficiently to affect host range expression. In addition, we tested the assumption that a state-dependent shift in motivation to oviposit on the target host will translate to a similar change in responsiveness to lower-ranked host species. Three-day-old and 10-day-old females of the candidate classical biological control agent, Diadromus pulchellus, were offered 12 non-target species of varying relatedness to the target pest, Acrolepiopsis assectella, in a series of no-choice and choice oviposition trials. Younger D. pulchellus females had previously demonstrated greater motivation to oviposit in the target pest and were, therefore, predicted to express a broader host range than older females. Parasitoid age had a minor effect on host range expression that was contrary to expectations. Older females more readily attacked one of the non-target species in no-choice tests and inflicted higher mortality in one of the choice tests. Ultimately however, young and old parasitoids still attacked the same four non-target species and their offspring emerged from the same three. There was an interaction between the effects of parasitoid condition and experimental design on responsiveness to low-ranked hosts: increasing non-target density in choice tests significantly altered attack rates by 10-day-old, but not by 3-day-old, parasitoids. The implications of these findings for host specificity testing depend largely on the specific aims of a host range assessment. Parasitoid state influenced the frequency of non-target attack but did not affect which non-target species were attacked. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction ⇑ Corresponding author. E-mail address: [email protected] (N. Cappuccino). http://dx.doi.org/10.1016/j.biocontrol.2014.07.005 1049-9644/Ó 2014 Elsevier Inc. All rights reserved.
Biological control is a process of managing pest organisms through the manipulation of species composition and abundance
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in targeted ecosystems. The modern practice of classical biological control, in which exotic agents are introduced to suppress exotic pests, typically involves a pre-introduction risk assessment of the proposed agent (Van Driesche, 2004; Hunt et al., 2008; Barratt et al., 2010). This evaluation is based primarily on an estimation of the prospective agent’s ecological host range (= the set of species used for reproduction in the field (sensu Nechols et al., 1992; Onstad and McManus, 1996)). While ecological host range can be measured directly through rigorous field surveys in the agent’s native range (e.g., Haye et al., 2005), a more common approach is to extrapolate a plausible approximation based on the agent’s fundamental host range (= the set of all species that can support development of the agent) in simple laboratory trials (Onstad and McManus, 1996). The latter method, however, is often criticised for exaggerating the ecological host range by inducing unnatural behaviours (Keller, 1999; Kidd and Jervis, 2005). This is because fundamental host range tests are typically run under artificial or semi-natural conditions and exclude the initial steps of the host search process. Despite these criticisms, many practitioners and regulators of classical biological control agree that it is desirable, at least in the early stages of host range assessment, to minimise the risk of underestimating host range (e.g., Buckingham and Bennett, 1998; Withers and Browne, 2004; van Lenteren et al., 2006). Thus, researchers have the daunting task of defining host range limits as accurately as possible without risking false-negative results (sensu Marohasy, 1998) that could result in the release of an agent that negatively affects the environment. Particular care must be taken to ensure that rejection of non-target species is not an artefact of the experimental design. There has been a growing theoretical interest in the importance of the physiological and informational state (i.e., energy state, life expectancy, mating status, etc.) of parasitoids in host acceptance studies (Roitberg, 2000). Since oviposition behaviour can be influenced by seemingly subtle factors (e.g., Roitberg et al., 1993; Goubault et al., 2005), it has been suggested that the condition of agents should be manipulated to reduce the risk of false-negative results. In theory, enhancing a parasitoid’s ‘‘motivation to oviposit’’ should lead it to express the widest possible host range (Withers and Browne, 2004). In practice, however, this does not appear to have been adopted in arthropod biological control programmes. This is illustrated by the fact that authors often neglect to describe the treatment of agents prior to testing. For instance, a review of the Materials and Methods from 86 host specificity tests on parasitoids (1984–2013) revealed that only 31 (37.2%) of them explicitly described all of the following five parasitoid conditions: mating status, nutritional status, age, host experience and parasitoid density (Jenner et al., unpublished results 2014). The effects of parasitoid state on host range expression have been investigated in only a handful of studies (e.g., Hare, 1996; Bjorksten and Hoffmann, 1998; Rousse et al., 2006), all of which have focused primarily on the impact of learning caused by previous host experience (informational state). It is now clear that both pre- and post-imaginal learning can have a strong influence on a parasitoid’s responsiveness to different hosts. In contrast, little effort has been made to explicitly test how host range is affected by changes in the physiological condition (i.e., age, egg load, mating and nutritional status, etc.) of the parasitoid. In this study, we tackled the question of whether a shift in motivation to oviposit in the target host induced by variation in physiological state will translate to a similar change in responsiveness to lower-ranked host species. As a model parasitoid-host system, we used the leek moth Acrolepiopsis assectella (Zeller) (Lepidoptera: Acrolepiidae) and its pupal parasitoid, Diadromus pulchellus Wesmael (Hymenoptera: Ichneumonidae). D. pulchellus is a solitary endoparasitoid that
attacks the pupal stage of A. assectella (Rousse, 1977). Mating takes place immediately after emergence (Rousse, 1973; Jenner et al., 2010). Labeyrie (1960) showed that the number of eggs laid per day increases until 6 days after female emergence when a maximum of 10–12 are laid. After 9 days the numbers fall to <3 per day by 14 days after emergence, and oviposition continues, but at decreasing rates, until death at 35 days. In the laboratory, mated female D. pulchellus laid an average of 78–98 eggs during their lifetime when offered 1–5 hosts per day (Labeyrie, 1964). Jenner et al. (2012) demonstrated that 3-day-old D. pulchellus females produced more offspring and killed more leek moth hosts than their 10-day-old siblings did. Based on these observations, we expected younger individuals to express a broader host range through more frequent attacks on non-target hosts (Withers and Browne, 2004). We conducted a thorough series of host specificity trials with D. pulchellus to delineate the boundaries of its host range and assess how parasitoid age would influence the outcome of such tests. The trials included both no-choice and choice assays to examine whether an age effect would be more noticeable when using particular experimental designs. 2. Materials and methods 2.1. Selection of non-target species The test list for assessing the host specificity of D. pulchellus is shown in Table 1. Each species included in the list fit at least one of the following selection criteria, derived from Kuhlmann et al. (2006): (1) phylogenetic affinity to target pest, (2) ecological similarity to target, (3), safeguard species (i.e., beneficial or rare), (4) morphological similarity to target and (5) known host of another Diadromus sp. parasitoid. 2.2. Rearing and preparation of D. pulchellus A laboratory colony of D. pulchellus was established with parasitoids collected in Switzerland in 2005 and was maintained continuously with field-collected specimens added each summer thereafter. The culture was maintained on leek moth hosts that were mass-reared on potted leeks in the laboratory. Leek moth pupae parasitised by D. pulchellus were stored in clean Petri dishes without host plant material for the duration of development. Newly emerged D. pulchellus females allocated to experiments were separated and housed in Petri dishes (9 cm width 2 cm depth) with two males until used in non-target trials. Each female was randomly assigned to the ‘‘young’’ or ‘‘old’’ treatment and then held in their Petri dishes for 3 or 10 days, respectively, prior to testing. As sugar-fed females have higher oviposition rates (Jenner et al., 2012), each Petri dish contained a cotton bud soaked in 20% sucrose solution, which was refreshed every second day. Jenner et al. (2012) also demonstrated that previous host experience can affect subsequent interactions between D. pulchellus and its hosts. In host specificity testing, there is a risk that agents will show a bias towards the target or rearing host due to pre- or post-emergence conditioning (Withers and Browne, 2004). To minimise the risk of associative learning prior to testing, only naïve parasitoids (without previous oviposition experience) were used in the nontarget trials. All parasitoids emerged from their hosts in the absence of plant material and were stored in clean Petri dishes with no access to hosts. To further reduce the chance of an experience-induced bias for the rearing host’s food plant, all nontarget and leek moth hosts in the Petri dish no-choice and choice tests were presented, in their cocoons when applicable, on an inert surface without the host plant (Withers and Browne, 2004). All
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Table 1 List of non-targets and their respective host plants for Diadromus pulchellus host specificity testing. Insect species (family)
Host plant (family)
Target Acrolepiopsis assectella (Zeller) (Acrolepiidae) Allium porrum (Linnaeus) (Alliaceae)
Criteria for selection
–
Non-targets Plutella xylostella (Linnaeus) (Plutellidae)
Brassica napus (Linnaeus) (Brassicaceae) Phylogenetic affinity ecological similarity morphological similarity host of a Diadromus sp. Plutella porrectella (Linnaeus) (Plutellidae) Hesperis matronalis (Linnaeus) Phylogenetic affinity ecological similarity morphological similarity (Brassicaceae) Acrolepiopsis incertella (Chambers) Smilax tamnoides (Linnaeus) Phylogenetic affinity safeguard species morphological similarity (Acrolepiidae) (Smilacaceae) Ypsolopha dentella (Fabricius) (Ypsolophidae) Lonicera tatarica (Linnaeus) Phylogenetic affinity morphological similarity (Caprifoliaceae) Yponomeuta padella (Linnaeus) Crataegus monogyna (Jacquin) Phylogenetic affinity (Yponomeutidae) (Rosaceae) Yponomeuta cagnagella (Huebner) Euonymus europaeus (Linnaeus) Phylogenetic affinity (Yponomeutidae) (Celastraceae) Athrips mouffetella (Linnaeus) (Gelechiidae) Lonicera tatarica (Linnaeus) Morphological affinity (Caprifoliaceae) Lobesia botrana (Denis & Schiff.) (Tortricidae) Vitis vinifera (Linnaeus) (Vitaceae) Host of a Diadromus sp. Morphological similarity Plodia interpunctella (Huebner) (Pyralidae) Stored grain mixa Spodoptera exigua (Huebner) (Noctuidae) Brassica oleracea (Linnaeus) Ecological similarity (Brassicaceae) Mamestra configurata Walker (Noctuidae) Brassica napus (Linnaeus) (Brassicaceae) Ecological similarity Pieris rapae (Linnaeus) (Pieridae) Brassica oleracea (Linnaeus) Ecological similarity (Brassicaceae) a
The stored grain mix consists of flour, almonds and rice.
experiments and insect rearing and storage were conducted at 22 ± 2 °C, 16L:8D.
2.3. Rearing of non-target hosts The non-targets tested with D. pulchellus were obtained from different sources. Four species were acquired from continuous rearing at commercial insectaries or research facilities as follows: Lobesia botrana (Denis and Schiffermüller) from Agroscope Changins-Wädenswil (Nyon, Switzerland), Spodoptera exigua (Hübner) from Bio-Serv (Frenchtown, NJ, USA), and Plutella xylostella (L.) and Mamestra configurata Walker from the Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada (Ottawa, ON, Canada). Temporary cultures of the remaining eight species were established using field-collected larvae of Plutella porrectella (L.), Acrolepiopsis incertella (Chambers), Ypsolopha dentella (Fabricius), Yponomeuta padella (L.), Yponomeuta cagnagella (Hübner) and adults of Plodia interpunctella (Hübner) and Pieris rapae (L.). All non-target species were reared in the laboratory on natural host plant material (see Table 1), with the exception of P. interpunctella, which was reared on a mixture of wheat flour, almonds and rice. The cultures were monitored daily to collect all newly formed pupae for immediate use in host fidelity trials. All target and non-target pupae used in experiments had pupated within the previous 24 h.
2.4. Simultaneous, no-choice, black-box test All 12 non-target species were exposed to D. pulchellus females in small arena, no-choice tests. These were also black-box trials in that the interaction between parasitoid and host was not closely monitored, the primary objective being to measure non-target mortality and parasitoid offspring emergence. Trials were, however, occasionally observed for a short period after introduction of the parasitoids to verify that the females were actively searching for hosts (van Lenteren et al., 2006). For each non-target trial, a single, mated, naïve D. pulchellus female was released into a clean
Petri dish (9 cm 2 cm) containing a single, 1-day-old non-target pupa and a cotton wick soaked with a 20% sucrose solution. Positive and negative controls were run simultaneously for each nontarget trial. Positive controls (to ensure that lack of attack is not due to problems with parasitoid quality or unfavourable conditions) were run by releasing another mated parasitoid of the same age into a second Petri dish with a 1-day-old pupa of the target, leek moth. Negative controls (to determine whether non-target death was caused by a factor other than parasitism) consisted of a non-target pupa prepared in the same way as described above but with no parasitoid added to the arena. All hosts were presented to parasitoids in the absence of host plant material; however, as the hosts had not been washed, they likely carried the scent of their host plants. The experiment was run for 24 h to provide sufficient time for each parasitoid to attack the pupa in its Petri dish and to control for a possible ‘‘time-of-day effect’’. This long exposure time should also increase the likelihood of non-target acceptance due to the progressive decrease in acceptance threshold that occurs during a period of host deprivation (Browne and Withers, 2002). At the end of the trial period, parasitoids were discarded and the pupae were stored in labelled Petri dishes to observe development. For every non-target species, a minimum of 100 individuals were tested, 50 with 3-day-old parasitoid females and 50 with 10-day-old females. Attack on each non-target species and the leek moth controls were estimated based on percent of parasitoid and moth emergence as well as the incidence of premature death of the host pupae. The specific response variables measured were host mortality (i.e. failure of a non-target adult to emerge due to physical damage caused by the parasitoid, complete or partial development of a parasitoid larva, or poor health of the pupa) and successful emergence of parasitoid progeny in the non-target and target hosts. A non-target species was deemed ‘‘acceptable’’ for oviposition if parasitoids were observed attacking the hosts or if the number of dead pupae was greater in the parasitoid treatments than the negative controls. A species was considered ‘‘suitable’’ if the agent was able to complete development to the adult stage.
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2.5. Choice tests between A. assectella and P. xylostella Following no-choice tests with all 12 non-target species, trials were conducted in which D. pulchellus was given a choice between leek moth and the non-target P. xylostella. This non-target species was used because it was already known to be a suitable host for D. pulchellus (Thibout, 1988). Thus, choice tests of increasing complexity (see below) were run to determine whether young and old parasitoids still attack the non-target in the presence of leek moth and to explore how the experimental design can affect the agent’s acceptance of a suitable non-target host. 2.5.1. Direct choice test in vial The initial choice test was conducted using small plastic vials (3 cm diameter 10 cm height) with target and non-target hosts placed only 1–2 mm apart. On the floor of each vial, one leek moth and one P. xylostella cocoon were fixed side-by-side with fine insect pins. The objective of this test was to measure the frequency of attacks on the target and non-target hosts when the parasitoid is able to detect both simultaneously (a true choice test). A single, mated, naïve parasitoid aged 3 or 10 days was introduced into the vial, which was sealed with a sponge plug. Each parasitoid was observed until it had discovered the pupae and made a ‘‘choice’’, defined as showing oviposition behaviour (bending of abdomen) or coming to rest on one host for >1 min. These two behaviours only occurred following a period of cocoon inspection. For 3-day-old and 10-day-old groups, each of the 30 replicates was terminated once a choice had been made and, in all cases, this occurred within 5 min. 2.5.2. Petri dish choice test (1:1) Two pupae (one leek moth and one P. xylostella) were placed in a Petri dish (9 cm 2 cm) along with a cotton bud soaked with 20% sucrose solution. In this experiment, the pupae were placed approximately 5 cm apart, always >1 cm away from the edge of the arena, and were anchored in place by pinning their cocoons to a small piece of filter paper. The first aim of this test was to observe how often parasitoids chose P. xylostella when there was a leek moth host available. As in the previous experiment, a single parasitoid was released into the arena and observed until a choice had been made. Unlike the previous experiment, however, the parasitoid was left in the arena with its hosts for 24 h. Formal observation ended once the first host had been attacked (typically within 5 min), therefore nearly the entire trial period was unmonitored. Following each of the 30 replicates, the pupae were stored in labelled Petri dishes and monitored to record host mortality and parasitoid emergence. 2.5.3. Petri dish choice test (1:4) Four pupae (one leek moth and three P. xylostella) were placed in a Petri dish as described in the preceding experiment. For this test, the pupae were randomly spread across the floor of the arena, spaced at least 2 cm apart and away from the dish edge. This experiment was a test of the prediction by Withers and Browne (2004) that presenting greater numbers of non-target hosts in choice tests could lead to increased acceptance rates of those species. Again, a single agent was introduced into the arena, observed until it had attacked its first host, and then left in the arena for 24 h. Afterwards, the pupae were stored as described above to obtain data on the target and non-target host mortalities and D. pulchellus emergence. 2.6. Data analysis All statistical analyses were carried out on arcsine square root transformed data using SPSS version 14.0 (SPSS Inc. (2005)). For
no-choice experiments, the chi-square test for association was used to test the null hypothesis that D. pulchellus age (3 vs. 10 days) had no effect on the number of non-target hosts killed or on the number of non-targets yielding healthy parasitoid offspring. The same test was used to assess whether there were differences in host mortality and progeny production for each non-target species compared to the target host. For non-targets from which no D. pulchellus offspring emerged, chi-square tests were used to determine whether non-target mortality was greater in the presence of parasitoids than in the negative controls. Since positive controls, using both young and old parasitoids on leek moth, were run for each non-target species tested, there were 12 independent samples of the impact that these parasitoids had on the target pest. A paired t-test was used to determine whether parasitoid age affected the rate of host mortality and progeny production. Chi-square tests were also used for all analyses involving data from the Petri dish choice tests with leek moth and P. xylostella. We used Bonferroni-corrected a-levels whenever multiple chisquare tests were conducted to test a given hypothesis. 3. Results 3.1. Simultaneous, no-choice, black-box test Of the 12 non-target species offered to D. pulchellus females in no-choice tests, only three (A. incertella, P. xylostella and P. porrectella) proved to be suitable for parasitoid development. When parasitoids encountered these non-target hosts, they were almost always arrested by contact with the cocoons. One additional nontarget, Y. dentella, was occasionally seen to elicit an oviposition response in D. pulchellus, however these attacks did not affect the survival of Y. dentella. In contrast, there were no observations of any strong response to the other eight non-targets. Parasitoid age did not have a significant effect on non-target mortality or parasitoid offspring emergence for any of the three suitable non-targets (P > 0.05 in all cases). The frequency with which D. pulchellus killed the non-targets A. incertella and P. xylostella was not significantly different from the frequency with which it killed its host, A. assectella. The non-target P. porrectella, however, was killed significantly less frequently than A. assectella, by both 3d-old and 10-d-old D. pulchellus (Fig. 1). When percent parasitoid emergence is considered, again the only significant differences were between P. porrectella and leek moth for both age treatments (Fig. 2). No difference in percent parasitoid emergence was detected for either age treatment with A. incertella or P. xylostella. Comparing the attack rates on leek moth by young and old parasitoids in the positive controls suggests that the older females have a higher motivation to oviposit. Host mortality was significantly higher in the old treatment (87.7 ± 1.8%; mean ± SE) than the young treatment (80.2 ± 1.4%) (t = 3.289; df = 11; P = 0.003). Since host quality was standardised among treatments, the higher frequency of host mortality with 10-day-old females is due to more frequent and/or more aggressive attacks by these parasitoids. An analysis of progeny production by young (47.3 ± 4.1%) and old (43.2 ± 3.2%) females failed to detect an age effect (t = 0.769; df = 11; P = 0.450). 3.2. Choice tests between A. assectella and P. xylostella 3.2.1. Direct choice test in vial When parasitoids were presented with a leek moth pupa next to a P. xylostella pupa, there was a very strong preference for the target species. Although both host species stimulated intense inspection by D. pulchellus, out of 30 trials, only two females – one 3- and one 10-day-old – chose P. xylostella by initiating ovipo-
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Fig. 1. Percent mortality (i.e., failing to yield a moth adult) of non-target and target hosts by (a) 3-day-old and (b) 10-day-old Diadromus pulchellus females in no-choice trials. Non-target species shown are Acrolepiopsis incertella, Plutella xylostella. and Plutella porrectella, while the target is Acrolepiopsis assectella. The number of trials for each treatment is shown in parentheses beneath the columns. ⁄P < 0.0016.
sition on this host first. However, in all cases where the parasitoids were first arrested by P. xylostella, they switched to the leek moth host once their antennae contacted its cocoon. In the two trials where D. pulchellus ‘‘chose’’ P. xylostella, the parasitoids actually abandoned the leek moth host before ovipositing and moved over to the non-target. 3.2.2. Petri dish choice test (1:1) When D. pulchellus females were introduced into a small arena containing one leek moth and one P. xylostella pupa separated by 5 cm, there was no significant influence of parasitoid age on the first host species attacked, although young parasitoids showed a slight preference for leek moth (v2 = 2.925; P = 0.087) compared to old parasitoids (v2 = 1.569; P = 0.210). Leek moth was killed in a significantly greater% of the trials than P. xylostella (Fig. 3a) and was significantly more likely to have been successfully parasitized by D. pulchellus, resulting in the emergence of parasitoid offspring (Fig. 4a). No age effect was detected for the number of P. xylostella hosts killed (v2 = 0.000; P = 1.000) or parasitoid emergence (v2 = 0.287; P = 0.592). 3.2.3. Petri dish choice test (1:4) Increasing the non-target to target ratio in the choice test appeared to affect only the 10-day-old parasitoids. Again, there was no significant age effect on the first host attacked; P. xylostella
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Fig. 2. Percent of no-choice trials in which parasitoids emerged from non-target and target hosts following exposure to (a) 3-day-old and (b) 10-day-old Diadromus pulchellus females. Non-target species shown are Acrolepiopsis incertella, Plutella xylostella and Plutella porrectella, while the target is Acrolepiopsis assectella. The number of trials for each non-target is shown in parentheses beneath the columns. ⁄ P < 0.0016.
was as likely as leek moth to be the first host attacked by both young (v2 = 0.939; P = 0.333) and old (v2 = 0.067; P = 0.796) parasitoids. As in the previous experiment, leek moth hosts exposed to young parasitoids were significantly more likely to be killed (Fig. 3b; v2 = 7.500; P = 0.006) and to yield D. pulchellus offspring (Fig. 4b; v2 = 10.335; P = 0.001) than were P. xylostella hosts, even though a higher density of the non-target hosts was available. In contrast, old females were equally likely to kill (v2 = 1.964; P = 0.161) and produce progeny (v2 = 0.000; P = 1.000) in the non-target when it was more abundant than leek moth. Further analyses comparing P. xylostella mortality between the 1:1 and 1:4 host ratio experiments confirm that 10-d-old D. pulchellus females were less host specific under higher non-target densities (v2 = 7.937; P = 0.005). Production of parasitoid progeny showed a similar pattern but was not statistically significant at the Bonferroni-corrected a-value of 0.0125 (v2 = 4.286; P = 0.038). No differences were found for the 3-day-old parasitoid treatments (host mortality: v2 = 0.067; P = 0.796; progeny production: v2 = 0.069; P = 0.793). Due to the probable enhanced motivation of older parasitoids to attack P. xylostella pupae in the 1:4 experiment, these parasitoids killed non-targets significantly more often than young ones did (v2 = 9.320; P = 0.002). There was, however, no difference in production of D. pulchellus progeny from P. xylostella hosts (v2 = 1.669; P = 0.196).
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Fig. 3. Percentage of Petri dish choice trials in which at least one non-target or target host was killed when a 3- or 10-day-old Diadromus pulchellus female was offered (a) one Acrolepiopsis assectella pupa with one Plutella xylostella pupa or (b) one A. assectella pupa with four P. xylostella pupae. ⁄P < 0.0125.
4. Discussion Host specificity has become a catch phrase in the field of classical arthropod biological control now that several countries require introduced agents to have relatively restricted host ranges (Kairo et al., 2003; Hunt et al., 2008; Barratt et al., 2010). Without reliable specificity tests, it is possible that safe agents could be rejected unnecessarily or that agents deemed suitable for release could negatively affect non-target populations. Because of the urgent need for reliable host range tests, efforts are ongoing to refine existing experimental protocols (Van Driesche and Reardon, 2004; Bigler et al., 2006). For example, experimental design has been a very important topic as it is well-known to affect the probability of non-target acceptance by agents (Sands and Van Driesche, 2000). The current study was a unique test of the importance of a parasitoid’s physiological state for the outcome of host specificity tests. We found that the age of D. pulchellus parasitoids did have a minor effect on performance in the different experiments. For instance, in the no-choice tests, older females killed the leek moth hosts more often in the positive controls and were also more likely to occasionally attack the unsuitable non-target, Y. dentella, upon initial contact. Moreover, the older parasitoids were more likely to kill P. xylostella hosts in the choice tests with one leek moth and four P. xylostella pupae. However, despite the slightly greater aggression and higher attack rates by older females, parasitoid age had no actual impact on host range expression. Young and old parasitoids still attacked the same four non-target species and their offspring emerged from the same three.
Fig. 4. Percentage of Petri dish choice trials in which at least one non-target or target host yielded parasitoid offspring when a 3- or 10-day-old Diadromus pulchellus female was offered (a) one Acrolepiopsis assectella pupa with one Plutella xylostella pupa or (b) one A. assectella pupa with four P. xylostella pupae. ⁄P < 0.0125.
The importance of parasitoid state in host specificity testing may depend on the test design. Our study revealed a potential interaction between the effects of host density and parasitoid condition on responsiveness to low-ranked hosts: increasing non-target density in choice tests substantially altered attack rates by 10day-old, but not by 3-day-old, parasitoids. Furthermore, it seems that the influence of parasitoid state on test results may depend on the specific response variables to be measured. For instance, while most host range assessments deal with the final stage of the foraging process (e.g., host acceptance), some studies may address earlier stages (e.g., habitat preference and responsiveness to odours) (Kitt and Keller, 1998). The effects of physiological state could therefore be highly variable since there is no evidence that responsiveness to odours and motivation to oviposit would be similarly affected by the physiological state of the forager. What are the implications of these findings for host specificity testing? The answer to this depends largely on the objectives of a host range assessment itself. If a researcher is simply interested in knowing whether a parasitoid can attack and develop on a particular non-target, then a single successful parasitism is sufficient to formulate a positive conclusion. In this case, the consequences of variable parasitoid state should be less important, provided that the agents are at least in a state that renders them capable of oviposition. It is probably of greater importance to ensure that acceptance trials are adequately replicated. Due to inaccessibility of certain non-targets or difficulty in rearing them, sample sizes are sometimes disturbingly small (e.g., Rogers and Potter, 2004; Goldson et al., 2005). When only a few individuals have been tested and there is no evidence of attack, the risk of obtaining false-negative results is high (Hoffmeister et al., 2006).
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On the other hand, a researcher may wish to obtain further data (e.g., frequency of attack) on the interaction between the agent and a non-target species in order to predict populationlevel consequences. In this case, parasitoid state could be of greater importance. Fig. 4b illustrates how choice tests run with 3-day-old parasitoids would lead one to conclude that the nontarget, P. xylostella, is significantly less preferred than leek moth. In contrast, the same experiment run with 10-day-old parasitoids would suggest that the non-target is equally likely as leek moth to be attacked. This difference in the laboratory would translate to a significant difference when extrapolated to the population scale. Thus, careful attention should be given to the physiological state of the agents when more than a simple yes/ no response is needed. While recognising the impact of parasitoid state on host range testing is an important refinement, the most significant obstacle is to determine how to manipulate an agent in order to enhance its motivation to oviposit. Due to operational constraints, it is not feasible for biological control practitioners to conduct meticulous experiments to identify the conditions necessary to enhance motivation to oviposit for every prospective agent. Withers and Browne (2004) therefore suggested several physiological and informational parasitoid states that should increase desire to oviposit and reduce the risk of false-negative results. These guidelines were based on foraging theory and drew supporting examples from empirical studies. However, with some exceptions (e.g., Bjorksten and Hoffmann, 1998; Rousse et al., 2006), most of the previous work on how parasitoid state affects foraging behaviour has investigated parasitoids interacting with their common hosts. Therefore, the recommendations of Withers and Browne (2004) are built on the premise that parasitoids with an enhanced readiness to oviposit in the target host should also be more willing to accept unfamiliar hosts and thus exhibit a wider host range. Our results give weak support for one of Withers and Browne’s specific predictions that older parasitoids would display a broader host range due to their perception of reduced life expectancy. Although we detected no age-effect in the no-choice tests, 10day-old D. pulchellus females were more willing than their 3-dayold siblings to attack non-target hosts in choice tests. It is interesting to note, however, that while this outcome matches predictions derived from foraging theory, it is contrary to our expectations from earlier experiments with this parasitoid and its common host. Jenner et al. (2012) demonstrated that 3-day-old D. pulchellus females killed more leek moth hosts and produced more offspring than 10-day-old females. Thus, the younger parasitoids had been deemed to have a higher motivation to oviposit and were expected to exhibit a broader host range. This illustrates that a researcher may not be able to identify specific parasitoid states that will maximise host range expression based on observations of state-dependent responsiveness to the target host. In summary, parasitoid physiological state can influence non-target host acceptance. However, due to the inconsistent effects that physiological state can have on parasitoid behaviour, a generalised set of guidelines for enhancing motivation to oviposit may not be appropriate for biological control agent evaluation. Nonetheless, this study highlights the importance of life history data for designing effective experiments. The degree to which parasitoid condition is manipulated prior to experimentation will ultimately depend on the depth of understanding of the parasitoid’s reproductive behaviour. For instance, knowing the duration of a pre-reproductive phase or the time of highest fecundity is useful for ensuring that agents are capable of performing the desired behaviours. Regardless of the benefits of manipulating parasitoid state, we suggest that thorough replication of non-target trials will remain the simplest way to minimise false-negative results.
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Acknowledgments We thank Richard Casagrande (University of Rhode Island) and John Arnason (University of Ottawa) for their useful comments on the manuscript. This work was supported by a Canada Graduate Scholarship from the Natural Sciences and Engineering Research Council of Canada and the Agriculture and Agri-Food Canada Pest Management Centre Improving Farming Systems and Practices Initiative Project PRR03-360. References Barratt, B.I.P., Howarth, F.G., Withers, T.M., Kean, J.M., Ridley, G.S., 2010. Progress in risk assessment for classical biological control. Biol. Control 52, 245–254. Bigler, F., Babendreier, D., Kuhlmann, U. (Eds.), 2006. Environmental Impact of Invertebrates for Biological Control of Arthropods: Methods and Risk Assessment. CABI Publishing, Wallingford, UK. Bjorksten, T.A., Hoffmann, A.A., 1998. Separating the effects of experience, size, egg load, and genotype on host response in Trichogramma (Hymenoptera: Trichogrammatidae). J. Insect Behav. 11, 129–148. 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Contents lists available at ScienceDirect
Biological Control journal homepage: www.elsevier.com/locate/ybcon
Does parasitoid state affect host range expression? W.H. Jenner a, U. Kuhlmann a, J.H. Miall b, N. Cappuccino c,⇑, P.G. Mason b a b c
CABI Europe – Switzerland, 1 Rue des Grillons, Delémont CH-2800, Switzerland Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada
h i g h l i g h t s 3-d-old & 10-d-old Diadromus
pulchellus were offered 12 non-targets in no-choice & choice trials. 3-d-old & 10-d-old parasitoids attacked four non-target species & offspring emerged from three. Increasing non-target density altered attack by 10-day-old, but not 3-dayold, parasitoids. Parasitoid state influenced frequency of non-target attack but did not affect species attacked.
a r t i c l e
i n f o
Article history: Received 4 March 2014 Accepted 14 July 2014 Available online 24 July 2014 Keywords: Acrolepiopsis assectella Classical biological control Diadromus pulchellus Experimental design Host specificity Non-target Physiological state
g r a p h i c a l a b s t r a c t Pupal parasitoid Diadromus pulchellus on leek moth hosts and non-target species or
or 3-d-old
10-d-old
14 non-target host species in separate no-choice tests
• No effect of parasitoid age on acceptance or mortality of non-target hosts.
3-d-old
One host pupa plus one nontarget pupa in choice test
10-d-old
or
One host pupa plus four nontarget pupae in choice test
• Aack rates by 3-d-old wasps did not depend on non-target density. • 10-d-old wasps were more likely to accept the non-target at higher densies.
a b s t r a c t The pre-release risk assessment of parasitoids for classical biological control generally involves nontarget testing to define the agent’s host range. To ensure that no suitable host species are falsely rejected in these tests, it has been suggested that the physiological and informational state of parasitoids be manipulated to enhance their ‘‘motivation to oviposit’’. However, the effects of such factors on host acceptance are not consistent across parasitoid species, making it laborious to identify the conditions necessary to maximise host acceptance. Our objective was to determine whether changes in parasitoid state could alter host acceptance behaviour sufficiently to affect host range expression. In addition, we tested the assumption that a state-dependent shift in motivation to oviposit on the target host will translate to a similar change in responsiveness to lower-ranked host species. Three-day-old and 10-day-old females of the candidate classical biological control agent, Diadromus pulchellus, were offered 12 non-target species of varying relatedness to the target pest, Acrolepiopsis assectella, in a series of no-choice and choice oviposition trials. Younger D. pulchellus females had previously demonstrated greater motivation to oviposit in the target pest and were, therefore, predicted to express a broader host range than older females. Parasitoid age had a minor effect on host range expression that was contrary to expectations. Older females more readily attacked one of the non-target species in no-choice tests and inflicted higher mortality in one of the choice tests. Ultimately however, young and old parasitoids still attacked the same four non-target species and their offspring emerged from the same three. There was an interaction between the effects of parasitoid condition and experimental design on responsiveness to low-ranked hosts: increasing non-target density in choice tests significantly altered attack rates by 10-day-old, but not by 3-day-old, parasitoids. The implications of these findings for host specificity testing depend largely on the specific aims of a host range assessment. Parasitoid state influenced the frequency of non-target attack but did not affect which non-target species were attacked. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction ⇑ Corresponding author. E-mail address: [email protected] (N. Cappuccino). http://dx.doi.org/10.1016/j.biocontrol.2014.07.005 1049-9644/Ó 2014 Elsevier Inc. All rights reserved.
Biological control is a process of managing pest organisms through the manipulation of species composition and abundance
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in targeted ecosystems. The modern practice of classical biological control, in which exotic agents are introduced to suppress exotic pests, typically involves a pre-introduction risk assessment of the proposed agent (Van Driesche, 2004; Hunt et al., 2008; Barratt et al., 2010). This evaluation is based primarily on an estimation of the prospective agent’s ecological host range (= the set of species used for reproduction in the field (sensu Nechols et al., 1992; Onstad and McManus, 1996)). While ecological host range can be measured directly through rigorous field surveys in the agent’s native range (e.g., Haye et al., 2005), a more common approach is to extrapolate a plausible approximation based on the agent’s fundamental host range (= the set of all species that can support development of the agent) in simple laboratory trials (Onstad and McManus, 1996). The latter method, however, is often criticised for exaggerating the ecological host range by inducing unnatural behaviours (Keller, 1999; Kidd and Jervis, 2005). This is because fundamental host range tests are typically run under artificial or semi-natural conditions and exclude the initial steps of the host search process. Despite these criticisms, many practitioners and regulators of classical biological control agree that it is desirable, at least in the early stages of host range assessment, to minimise the risk of underestimating host range (e.g., Buckingham and Bennett, 1998; Withers and Browne, 2004; van Lenteren et al., 2006). Thus, researchers have the daunting task of defining host range limits as accurately as possible without risking false-negative results (sensu Marohasy, 1998) that could result in the release of an agent that negatively affects the environment. Particular care must be taken to ensure that rejection of non-target species is not an artefact of the experimental design. There has been a growing theoretical interest in the importance of the physiological and informational state (i.e., energy state, life expectancy, mating status, etc.) of parasitoids in host acceptance studies (Roitberg, 2000). Since oviposition behaviour can be influenced by seemingly subtle factors (e.g., Roitberg et al., 1993; Goubault et al., 2005), it has been suggested that the condition of agents should be manipulated to reduce the risk of false-negative results. In theory, enhancing a parasitoid’s ‘‘motivation to oviposit’’ should lead it to express the widest possible host range (Withers and Browne, 2004). In practice, however, this does not appear to have been adopted in arthropod biological control programmes. This is illustrated by the fact that authors often neglect to describe the treatment of agents prior to testing. For instance, a review of the Materials and Methods from 86 host specificity tests on parasitoids (1984–2013) revealed that only 31 (37.2%) of them explicitly described all of the following five parasitoid conditions: mating status, nutritional status, age, host experience and parasitoid density (Jenner et al., unpublished results 2014). The effects of parasitoid state on host range expression have been investigated in only a handful of studies (e.g., Hare, 1996; Bjorksten and Hoffmann, 1998; Rousse et al., 2006), all of which have focused primarily on the impact of learning caused by previous host experience (informational state). It is now clear that both pre- and post-imaginal learning can have a strong influence on a parasitoid’s responsiveness to different hosts. In contrast, little effort has been made to explicitly test how host range is affected by changes in the physiological condition (i.e., age, egg load, mating and nutritional status, etc.) of the parasitoid. In this study, we tackled the question of whether a shift in motivation to oviposit in the target host induced by variation in physiological state will translate to a similar change in responsiveness to lower-ranked host species. As a model parasitoid-host system, we used the leek moth Acrolepiopsis assectella (Zeller) (Lepidoptera: Acrolepiidae) and its pupal parasitoid, Diadromus pulchellus Wesmael (Hymenoptera: Ichneumonidae). D. pulchellus is a solitary endoparasitoid that
attacks the pupal stage of A. assectella (Rousse, 1977). Mating takes place immediately after emergence (Rousse, 1973; Jenner et al., 2010). Labeyrie (1960) showed that the number of eggs laid per day increases until 6 days after female emergence when a maximum of 10–12 are laid. After 9 days the numbers fall to <3 per day by 14 days after emergence, and oviposition continues, but at decreasing rates, until death at 35 days. In the laboratory, mated female D. pulchellus laid an average of 78–98 eggs during their lifetime when offered 1–5 hosts per day (Labeyrie, 1964). Jenner et al. (2012) demonstrated that 3-day-old D. pulchellus females produced more offspring and killed more leek moth hosts than their 10-day-old siblings did. Based on these observations, we expected younger individuals to express a broader host range through more frequent attacks on non-target hosts (Withers and Browne, 2004). We conducted a thorough series of host specificity trials with D. pulchellus to delineate the boundaries of its host range and assess how parasitoid age would influence the outcome of such tests. The trials included both no-choice and choice assays to examine whether an age effect would be more noticeable when using particular experimental designs. 2. Materials and methods 2.1. Selection of non-target species The test list for assessing the host specificity of D. pulchellus is shown in Table 1. Each species included in the list fit at least one of the following selection criteria, derived from Kuhlmann et al. (2006): (1) phylogenetic affinity to target pest, (2) ecological similarity to target, (3), safeguard species (i.e., beneficial or rare), (4) morphological similarity to target and (5) known host of another Diadromus sp. parasitoid. 2.2. Rearing and preparation of D. pulchellus A laboratory colony of D. pulchellus was established with parasitoids collected in Switzerland in 2005 and was maintained continuously with field-collected specimens added each summer thereafter. The culture was maintained on leek moth hosts that were mass-reared on potted leeks in the laboratory. Leek moth pupae parasitised by D. pulchellus were stored in clean Petri dishes without host plant material for the duration of development. Newly emerged D. pulchellus females allocated to experiments were separated and housed in Petri dishes (9 cm width 2 cm depth) with two males until used in non-target trials. Each female was randomly assigned to the ‘‘young’’ or ‘‘old’’ treatment and then held in their Petri dishes for 3 or 10 days, respectively, prior to testing. As sugar-fed females have higher oviposition rates (Jenner et al., 2012), each Petri dish contained a cotton bud soaked in 20% sucrose solution, which was refreshed every second day. Jenner et al. (2012) also demonstrated that previous host experience can affect subsequent interactions between D. pulchellus and its hosts. In host specificity testing, there is a risk that agents will show a bias towards the target or rearing host due to pre- or post-emergence conditioning (Withers and Browne, 2004). To minimise the risk of associative learning prior to testing, only naïve parasitoids (without previous oviposition experience) were used in the nontarget trials. All parasitoids emerged from their hosts in the absence of plant material and were stored in clean Petri dishes with no access to hosts. To further reduce the chance of an experience-induced bias for the rearing host’s food plant, all nontarget and leek moth hosts in the Petri dish no-choice and choice tests were presented, in their cocoons when applicable, on an inert surface without the host plant (Withers and Browne, 2004). All
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Table 1 List of non-targets and their respective host plants for Diadromus pulchellus host specificity testing. Insect species (family)
Host plant (family)
Target Acrolepiopsis assectella (Zeller) (Acrolepiidae) Allium porrum (Linnaeus) (Alliaceae)
Criteria for selection
–
Non-targets Plutella xylostella (Linnaeus) (Plutellidae)
Brassica napus (Linnaeus) (Brassicaceae) Phylogenetic affinity ecological similarity morphological similarity host of a Diadromus sp. Plutella porrectella (Linnaeus) (Plutellidae) Hesperis matronalis (Linnaeus) Phylogenetic affinity ecological similarity morphological similarity (Brassicaceae) Acrolepiopsis incertella (Chambers) Smilax tamnoides (Linnaeus) Phylogenetic affinity safeguard species morphological similarity (Acrolepiidae) (Smilacaceae) Ypsolopha dentella (Fabricius) (Ypsolophidae) Lonicera tatarica (Linnaeus) Phylogenetic affinity morphological similarity (Caprifoliaceae) Yponomeuta padella (Linnaeus) Crataegus monogyna (Jacquin) Phylogenetic affinity (Yponomeutidae) (Rosaceae) Yponomeuta cagnagella (Huebner) Euonymus europaeus (Linnaeus) Phylogenetic affinity (Yponomeutidae) (Celastraceae) Athrips mouffetella (Linnaeus) (Gelechiidae) Lonicera tatarica (Linnaeus) Morphological affinity (Caprifoliaceae) Lobesia botrana (Denis & Schiff.) (Tortricidae) Vitis vinifera (Linnaeus) (Vitaceae) Host of a Diadromus sp. Morphological similarity Plodia interpunctella (Huebner) (Pyralidae) Stored grain mixa Spodoptera exigua (Huebner) (Noctuidae) Brassica oleracea (Linnaeus) Ecological similarity (Brassicaceae) Mamestra configurata Walker (Noctuidae) Brassica napus (Linnaeus) (Brassicaceae) Ecological similarity Pieris rapae (Linnaeus) (Pieridae) Brassica oleracea (Linnaeus) Ecological similarity (Brassicaceae) a
The stored grain mix consists of flour, almonds and rice.
experiments and insect rearing and storage were conducted at 22 ± 2 °C, 16L:8D.
2.3. Rearing of non-target hosts The non-targets tested with D. pulchellus were obtained from different sources. Four species were acquired from continuous rearing at commercial insectaries or research facilities as follows: Lobesia botrana (Denis and Schiffermüller) from Agroscope Changins-Wädenswil (Nyon, Switzerland), Spodoptera exigua (Hübner) from Bio-Serv (Frenchtown, NJ, USA), and Plutella xylostella (L.) and Mamestra configurata Walker from the Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada (Ottawa, ON, Canada). Temporary cultures of the remaining eight species were established using field-collected larvae of Plutella porrectella (L.), Acrolepiopsis incertella (Chambers), Ypsolopha dentella (Fabricius), Yponomeuta padella (L.), Yponomeuta cagnagella (Hübner) and adults of Plodia interpunctella (Hübner) and Pieris rapae (L.). All non-target species were reared in the laboratory on natural host plant material (see Table 1), with the exception of P. interpunctella, which was reared on a mixture of wheat flour, almonds and rice. The cultures were monitored daily to collect all newly formed pupae for immediate use in host fidelity trials. All target and non-target pupae used in experiments had pupated within the previous 24 h.
2.4. Simultaneous, no-choice, black-box test All 12 non-target species were exposed to D. pulchellus females in small arena, no-choice tests. These were also black-box trials in that the interaction between parasitoid and host was not closely monitored, the primary objective being to measure non-target mortality and parasitoid offspring emergence. Trials were, however, occasionally observed for a short period after introduction of the parasitoids to verify that the females were actively searching for hosts (van Lenteren et al., 2006). For each non-target trial, a single, mated, naïve D. pulchellus female was released into a clean
Petri dish (9 cm 2 cm) containing a single, 1-day-old non-target pupa and a cotton wick soaked with a 20% sucrose solution. Positive and negative controls were run simultaneously for each nontarget trial. Positive controls (to ensure that lack of attack is not due to problems with parasitoid quality or unfavourable conditions) were run by releasing another mated parasitoid of the same age into a second Petri dish with a 1-day-old pupa of the target, leek moth. Negative controls (to determine whether non-target death was caused by a factor other than parasitism) consisted of a non-target pupa prepared in the same way as described above but with no parasitoid added to the arena. All hosts were presented to parasitoids in the absence of host plant material; however, as the hosts had not been washed, they likely carried the scent of their host plants. The experiment was run for 24 h to provide sufficient time for each parasitoid to attack the pupa in its Petri dish and to control for a possible ‘‘time-of-day effect’’. This long exposure time should also increase the likelihood of non-target acceptance due to the progressive decrease in acceptance threshold that occurs during a period of host deprivation (Browne and Withers, 2002). At the end of the trial period, parasitoids were discarded and the pupae were stored in labelled Petri dishes to observe development. For every non-target species, a minimum of 100 individuals were tested, 50 with 3-day-old parasitoid females and 50 with 10-day-old females. Attack on each non-target species and the leek moth controls were estimated based on percent of parasitoid and moth emergence as well as the incidence of premature death of the host pupae. The specific response variables measured were host mortality (i.e. failure of a non-target adult to emerge due to physical damage caused by the parasitoid, complete or partial development of a parasitoid larva, or poor health of the pupa) and successful emergence of parasitoid progeny in the non-target and target hosts. A non-target species was deemed ‘‘acceptable’’ for oviposition if parasitoids were observed attacking the hosts or if the number of dead pupae was greater in the parasitoid treatments than the negative controls. A species was considered ‘‘suitable’’ if the agent was able to complete development to the adult stage.
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2.5. Choice tests between A. assectella and P. xylostella Following no-choice tests with all 12 non-target species, trials were conducted in which D. pulchellus was given a choice between leek moth and the non-target P. xylostella. This non-target species was used because it was already known to be a suitable host for D. pulchellus (Thibout, 1988). Thus, choice tests of increasing complexity (see below) were run to determine whether young and old parasitoids still attack the non-target in the presence of leek moth and to explore how the experimental design can affect the agent’s acceptance of a suitable non-target host. 2.5.1. Direct choice test in vial The initial choice test was conducted using small plastic vials (3 cm diameter 10 cm height) with target and non-target hosts placed only 1–2 mm apart. On the floor of each vial, one leek moth and one P. xylostella cocoon were fixed side-by-side with fine insect pins. The objective of this test was to measure the frequency of attacks on the target and non-target hosts when the parasitoid is able to detect both simultaneously (a true choice test). A single, mated, naïve parasitoid aged 3 or 10 days was introduced into the vial, which was sealed with a sponge plug. Each parasitoid was observed until it had discovered the pupae and made a ‘‘choice’’, defined as showing oviposition behaviour (bending of abdomen) or coming to rest on one host for >1 min. These two behaviours only occurred following a period of cocoon inspection. For 3-day-old and 10-day-old groups, each of the 30 replicates was terminated once a choice had been made and, in all cases, this occurred within 5 min. 2.5.2. Petri dish choice test (1:1) Two pupae (one leek moth and one P. xylostella) were placed in a Petri dish (9 cm 2 cm) along with a cotton bud soaked with 20% sucrose solution. In this experiment, the pupae were placed approximately 5 cm apart, always >1 cm away from the edge of the arena, and were anchored in place by pinning their cocoons to a small piece of filter paper. The first aim of this test was to observe how often parasitoids chose P. xylostella when there was a leek moth host available. As in the previous experiment, a single parasitoid was released into the arena and observed until a choice had been made. Unlike the previous experiment, however, the parasitoid was left in the arena with its hosts for 24 h. Formal observation ended once the first host had been attacked (typically within 5 min), therefore nearly the entire trial period was unmonitored. Following each of the 30 replicates, the pupae were stored in labelled Petri dishes and monitored to record host mortality and parasitoid emergence. 2.5.3. Petri dish choice test (1:4) Four pupae (one leek moth and three P. xylostella) were placed in a Petri dish as described in the preceding experiment. For this test, the pupae were randomly spread across the floor of the arena, spaced at least 2 cm apart and away from the dish edge. This experiment was a test of the prediction by Withers and Browne (2004) that presenting greater numbers of non-target hosts in choice tests could lead to increased acceptance rates of those species. Again, a single agent was introduced into the arena, observed until it had attacked its first host, and then left in the arena for 24 h. Afterwards, the pupae were stored as described above to obtain data on the target and non-target host mortalities and D. pulchellus emergence. 2.6. Data analysis All statistical analyses were carried out on arcsine square root transformed data using SPSS version 14.0 (SPSS Inc. (2005)). For
no-choice experiments, the chi-square test for association was used to test the null hypothesis that D. pulchellus age (3 vs. 10 days) had no effect on the number of non-target hosts killed or on the number of non-targets yielding healthy parasitoid offspring. The same test was used to assess whether there were differences in host mortality and progeny production for each non-target species compared to the target host. For non-targets from which no D. pulchellus offspring emerged, chi-square tests were used to determine whether non-target mortality was greater in the presence of parasitoids than in the negative controls. Since positive controls, using both young and old parasitoids on leek moth, were run for each non-target species tested, there were 12 independent samples of the impact that these parasitoids had on the target pest. A paired t-test was used to determine whether parasitoid age affected the rate of host mortality and progeny production. Chi-square tests were also used for all analyses involving data from the Petri dish choice tests with leek moth and P. xylostella. We used Bonferroni-corrected a-levels whenever multiple chisquare tests were conducted to test a given hypothesis. 3. Results 3.1. Simultaneous, no-choice, black-box test Of the 12 non-target species offered to D. pulchellus females in no-choice tests, only three (A. incertella, P. xylostella and P. porrectella) proved to be suitable for parasitoid development. When parasitoids encountered these non-target hosts, they were almost always arrested by contact with the cocoons. One additional nontarget, Y. dentella, was occasionally seen to elicit an oviposition response in D. pulchellus, however these attacks did not affect the survival of Y. dentella. In contrast, there were no observations of any strong response to the other eight non-targets. Parasitoid age did not have a significant effect on non-target mortality or parasitoid offspring emergence for any of the three suitable non-targets (P > 0.05 in all cases). The frequency with which D. pulchellus killed the non-targets A. incertella and P. xylostella was not significantly different from the frequency with which it killed its host, A. assectella. The non-target P. porrectella, however, was killed significantly less frequently than A. assectella, by both 3d-old and 10-d-old D. pulchellus (Fig. 1). When percent parasitoid emergence is considered, again the only significant differences were between P. porrectella and leek moth for both age treatments (Fig. 2). No difference in percent parasitoid emergence was detected for either age treatment with A. incertella or P. xylostella. Comparing the attack rates on leek moth by young and old parasitoids in the positive controls suggests that the older females have a higher motivation to oviposit. Host mortality was significantly higher in the old treatment (87.7 ± 1.8%; mean ± SE) than the young treatment (80.2 ± 1.4%) (t = 3.289; df = 11; P = 0.003). Since host quality was standardised among treatments, the higher frequency of host mortality with 10-day-old females is due to more frequent and/or more aggressive attacks by these parasitoids. An analysis of progeny production by young (47.3 ± 4.1%) and old (43.2 ± 3.2%) females failed to detect an age effect (t = 0.769; df = 11; P = 0.450). 3.2. Choice tests between A. assectella and P. xylostella 3.2.1. Direct choice test in vial When parasitoids were presented with a leek moth pupa next to a P. xylostella pupa, there was a very strong preference for the target species. Although both host species stimulated intense inspection by D. pulchellus, out of 30 trials, only two females – one 3- and one 10-day-old – chose P. xylostella by initiating ovipo-
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Fig. 1. Percent mortality (i.e., failing to yield a moth adult) of non-target and target hosts by (a) 3-day-old and (b) 10-day-old Diadromus pulchellus females in no-choice trials. Non-target species shown are Acrolepiopsis incertella, Plutella xylostella. and Plutella porrectella, while the target is Acrolepiopsis assectella. The number of trials for each treatment is shown in parentheses beneath the columns. ⁄P < 0.0016.
sition on this host first. However, in all cases where the parasitoids were first arrested by P. xylostella, they switched to the leek moth host once their antennae contacted its cocoon. In the two trials where D. pulchellus ‘‘chose’’ P. xylostella, the parasitoids actually abandoned the leek moth host before ovipositing and moved over to the non-target. 3.2.2. Petri dish choice test (1:1) When D. pulchellus females were introduced into a small arena containing one leek moth and one P. xylostella pupa separated by 5 cm, there was no significant influence of parasitoid age on the first host species attacked, although young parasitoids showed a slight preference for leek moth (v2 = 2.925; P = 0.087) compared to old parasitoids (v2 = 1.569; P = 0.210). Leek moth was killed in a significantly greater% of the trials than P. xylostella (Fig. 3a) and was significantly more likely to have been successfully parasitized by D. pulchellus, resulting in the emergence of parasitoid offspring (Fig. 4a). No age effect was detected for the number of P. xylostella hosts killed (v2 = 0.000; P = 1.000) or parasitoid emergence (v2 = 0.287; P = 0.592). 3.2.3. Petri dish choice test (1:4) Increasing the non-target to target ratio in the choice test appeared to affect only the 10-day-old parasitoids. Again, there was no significant age effect on the first host attacked; P. xylostella
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Fig. 2. Percent of no-choice trials in which parasitoids emerged from non-target and target hosts following exposure to (a) 3-day-old and (b) 10-day-old Diadromus pulchellus females. Non-target species shown are Acrolepiopsis incertella, Plutella xylostella and Plutella porrectella, while the target is Acrolepiopsis assectella. The number of trials for each non-target is shown in parentheses beneath the columns. ⁄ P < 0.0016.
was as likely as leek moth to be the first host attacked by both young (v2 = 0.939; P = 0.333) and old (v2 = 0.067; P = 0.796) parasitoids. As in the previous experiment, leek moth hosts exposed to young parasitoids were significantly more likely to be killed (Fig. 3b; v2 = 7.500; P = 0.006) and to yield D. pulchellus offspring (Fig. 4b; v2 = 10.335; P = 0.001) than were P. xylostella hosts, even though a higher density of the non-target hosts was available. In contrast, old females were equally likely to kill (v2 = 1.964; P = 0.161) and produce progeny (v2 = 0.000; P = 1.000) in the non-target when it was more abundant than leek moth. Further analyses comparing P. xylostella mortality between the 1:1 and 1:4 host ratio experiments confirm that 10-d-old D. pulchellus females were less host specific under higher non-target densities (v2 = 7.937; P = 0.005). Production of parasitoid progeny showed a similar pattern but was not statistically significant at the Bonferroni-corrected a-value of 0.0125 (v2 = 4.286; P = 0.038). No differences were found for the 3-day-old parasitoid treatments (host mortality: v2 = 0.067; P = 0.796; progeny production: v2 = 0.069; P = 0.793). Due to the probable enhanced motivation of older parasitoids to attack P. xylostella pupae in the 1:4 experiment, these parasitoids killed non-targets significantly more often than young ones did (v2 = 9.320; P = 0.002). There was, however, no difference in production of D. pulchellus progeny from P. xylostella hosts (v2 = 1.669; P = 0.196).
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Fig. 3. Percentage of Petri dish choice trials in which at least one non-target or target host was killed when a 3- or 10-day-old Diadromus pulchellus female was offered (a) one Acrolepiopsis assectella pupa with one Plutella xylostella pupa or (b) one A. assectella pupa with four P. xylostella pupae. ⁄P < 0.0125.
4. Discussion Host specificity has become a catch phrase in the field of classical arthropod biological control now that several countries require introduced agents to have relatively restricted host ranges (Kairo et al., 2003; Hunt et al., 2008; Barratt et al., 2010). Without reliable specificity tests, it is possible that safe agents could be rejected unnecessarily or that agents deemed suitable for release could negatively affect non-target populations. Because of the urgent need for reliable host range tests, efforts are ongoing to refine existing experimental protocols (Van Driesche and Reardon, 2004; Bigler et al., 2006). For example, experimental design has been a very important topic as it is well-known to affect the probability of non-target acceptance by agents (Sands and Van Driesche, 2000). The current study was a unique test of the importance of a parasitoid’s physiological state for the outcome of host specificity tests. We found that the age of D. pulchellus parasitoids did have a minor effect on performance in the different experiments. For instance, in the no-choice tests, older females killed the leek moth hosts more often in the positive controls and were also more likely to occasionally attack the unsuitable non-target, Y. dentella, upon initial contact. Moreover, the older parasitoids were more likely to kill P. xylostella hosts in the choice tests with one leek moth and four P. xylostella pupae. However, despite the slightly greater aggression and higher attack rates by older females, parasitoid age had no actual impact on host range expression. Young and old parasitoids still attacked the same four non-target species and their offspring emerged from the same three.
Fig. 4. Percentage of Petri dish choice trials in which at least one non-target or target host yielded parasitoid offspring when a 3- or 10-day-old Diadromus pulchellus female was offered (a) one Acrolepiopsis assectella pupa with one Plutella xylostella pupa or (b) one A. assectella pupa with four P. xylostella pupae. ⁄P < 0.0125.
The importance of parasitoid state in host specificity testing may depend on the test design. Our study revealed a potential interaction between the effects of host density and parasitoid condition on responsiveness to low-ranked hosts: increasing non-target density in choice tests substantially altered attack rates by 10day-old, but not by 3-day-old, parasitoids. Furthermore, it seems that the influence of parasitoid state on test results may depend on the specific response variables to be measured. For instance, while most host range assessments deal with the final stage of the foraging process (e.g., host acceptance), some studies may address earlier stages (e.g., habitat preference and responsiveness to odours) (Kitt and Keller, 1998). The effects of physiological state could therefore be highly variable since there is no evidence that responsiveness to odours and motivation to oviposit would be similarly affected by the physiological state of the forager. What are the implications of these findings for host specificity testing? The answer to this depends largely on the objectives of a host range assessment itself. If a researcher is simply interested in knowing whether a parasitoid can attack and develop on a particular non-target, then a single successful parasitism is sufficient to formulate a positive conclusion. In this case, the consequences of variable parasitoid state should be less important, provided that the agents are at least in a state that renders them capable of oviposition. It is probably of greater importance to ensure that acceptance trials are adequately replicated. Due to inaccessibility of certain non-targets or difficulty in rearing them, sample sizes are sometimes disturbingly small (e.g., Rogers and Potter, 2004; Goldson et al., 2005). When only a few individuals have been tested and there is no evidence of attack, the risk of obtaining false-negative results is high (Hoffmeister et al., 2006).
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On the other hand, a researcher may wish to obtain further data (e.g., frequency of attack) on the interaction between the agent and a non-target species in order to predict populationlevel consequences. In this case, parasitoid state could be of greater importance. Fig. 4b illustrates how choice tests run with 3-day-old parasitoids would lead one to conclude that the nontarget, P. xylostella, is significantly less preferred than leek moth. In contrast, the same experiment run with 10-day-old parasitoids would suggest that the non-target is equally likely as leek moth to be attacked. This difference in the laboratory would translate to a significant difference when extrapolated to the population scale. Thus, careful attention should be given to the physiological state of the agents when more than a simple yes/ no response is needed. While recognising the impact of parasitoid state on host range testing is an important refinement, the most significant obstacle is to determine how to manipulate an agent in order to enhance its motivation to oviposit. Due to operational constraints, it is not feasible for biological control practitioners to conduct meticulous experiments to identify the conditions necessary to enhance motivation to oviposit for every prospective agent. Withers and Browne (2004) therefore suggested several physiological and informational parasitoid states that should increase desire to oviposit and reduce the risk of false-negative results. These guidelines were based on foraging theory and drew supporting examples from empirical studies. However, with some exceptions (e.g., Bjorksten and Hoffmann, 1998; Rousse et al., 2006), most of the previous work on how parasitoid state affects foraging behaviour has investigated parasitoids interacting with their common hosts. Therefore, the recommendations of Withers and Browne (2004) are built on the premise that parasitoids with an enhanced readiness to oviposit in the target host should also be more willing to accept unfamiliar hosts and thus exhibit a wider host range. Our results give weak support for one of Withers and Browne’s specific predictions that older parasitoids would display a broader host range due to their perception of reduced life expectancy. Although we detected no age-effect in the no-choice tests, 10day-old D. pulchellus females were more willing than their 3-dayold siblings to attack non-target hosts in choice tests. It is interesting to note, however, that while this outcome matches predictions derived from foraging theory, it is contrary to our expectations from earlier experiments with this parasitoid and its common host. Jenner et al. (2012) demonstrated that 3-day-old D. pulchellus females killed more leek moth hosts and produced more offspring than 10-day-old females. Thus, the younger parasitoids had been deemed to have a higher motivation to oviposit and were expected to exhibit a broader host range. This illustrates that a researcher may not be able to identify specific parasitoid states that will maximise host range expression based on observations of state-dependent responsiveness to the target host. In summary, parasitoid physiological state can influence non-target host acceptance. However, due to the inconsistent effects that physiological state can have on parasitoid behaviour, a generalised set of guidelines for enhancing motivation to oviposit may not be appropriate for biological control agent evaluation. Nonetheless, this study highlights the importance of life history data for designing effective experiments. The degree to which parasitoid condition is manipulated prior to experimentation will ultimately depend on the depth of understanding of the parasitoid’s reproductive behaviour. For instance, knowing the duration of a pre-reproductive phase or the time of highest fecundity is useful for ensuring that agents are capable of performing the desired behaviours. Regardless of the benefits of manipulating parasitoid state, we suggest that thorough replication of non-target trials will remain the simplest way to minimise false-negative results.
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