Single experience learning of host fruit selection by lepidopteran larvae

Physiology & Behavior 86 (2005) 168 – 175

Single experience learning of host fruit selection by lepidopteran larvae Maciej A. Pszczolkowski *, John J. Brown Department of Entomology, Washington State University, 166 FSHN, Pullman, WA 99163, USA Received 15 June 2005; accepted 6 July 2005

Abstract Neonate larvae of a lepidopteran, the codling moth (Cydia pomonella L.) search for their host fruit after hatch. The process of host searching is known to be activated by kairomones contained in host fruit volatiles, but the mechanism of actual selection and infestation of the fruit is unclear. Here we show that lepidopteran neonates can utilize single experience learning in selection and infestation of host apple. We found that the process of host fruit selection may be modified by single experience learning, namely preference induction or averse conditioning. Both types of learning were acquired within 3 h of training. Experience was retained for over 3 days in the case of averse conditioning. Preference induction, a form of learning specific to insects, is expected to produce rigid host preference lasting for days if not weeks, but in codling moth neonates this type of memory was retained only for 3 h. We speculate that conjunction of preference induction with short retention time and averse conditioning with long retention time provide an optimal adaptive strategy of host fruit selection for codling moth neonates. D 2005 Elsevier Inc. All rights reserved. Keywords: Insect; Codling moth; Cydia pomonella; Taste; Saccharin; Apple; Gingko

1. Introduction Adults of an important orchard pest, the codling moth, Cydia pomonellaL, (Lepidoptera: Tortricidae), have remarkable potential for migration. Mated and non-mated females can fly a distance of 15 km [1]. Flight parameters (duration, velocity and distance) are inheritable [2] and are subject to selection, which can be demonstrated in the field [3]. Codling moths can be found a distance from infested orchards, in areas isolated from them [4], which is well reflected by enzymatic differences among populations of this species [5]. Gravid females are not discriminative in their selection of foliage as oviposition substrate, and were reported to oviposit on foliage and feed in fruits of at least 8 hosts; apple, pear, quince, walnut, nectarine, peach, plum, and cherry [6 –8]. Apparently, trees of many species produce stimuli sufficient to induce oviposition in codling moth. In * Corresponding author. Department of Entomology, Kansas State University, 123 West Waters Hall, Manhattan, KS 66506, USA. Tel.: +1 785 532 4723; fax: +1 785 532 6232. E-mail address: [email protected] (M.A. Pszczolkowski). 0031-9384/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2005.07.007

addition, the preference of oviposition substrate by codling moth can be significantly modified by visual stimuli: there are data showing that in the field, codling moth females orient to tree silhouette rather than to apple odor [9]. Normally, neonate larvae infest the fruit (mostly the apple) and feed in it until their development is complete. Once having infested the fruit, the codling moth larva never leaves it until pupation [10,11]. However, in absence of suitable fruit, neonates of this species can also feed and develop to the next larval instar on foliage alone, including the leaves of the primary host, the apple, and that of secondary hosts such as the pear or the plum [10,12]. Because the fruit once infested is never left, the codling moth environment has high degree of within-generation predictability, but great potential for migration and nondiscriminative choice of oviposition substrate in the adults suggest that between-generation predictability of oviposition and food resources is low. It was once proposed [13] that in such a situation learning of host plant choice would be especially beneficial to an animal with discrete generations (high within-generation predictability combined with high between-generation predictability would favor

M.A. Pszczolkowski, J.J. Brown / Physiology & Behavior 86 (2005) 168 – 175

evolving a fixed response, whereas in any other combination learning would be useless). This model can be further extended by a hypothetical example of how learning may influence the direction of evolutionary change [14]. For instance, a gravid female butterfly having an excessive eggload becomes less selective in her choice of oviposition substrate [15,16]. It was proposed [14] that a female bearing a large egg-load and encountering a plant other than its normal host may lay eggs on the non-host, and that act of oviposition alone may provide positive reinforcement for this behavior. Persistent and reinforced oviposition on a novel host may increase the likelihood that some offspring will accept the new plant and develop on it [14]. We think that host fruit selection by the codling moth cannot be explained solely by persistent and reinforced oviposition behavior of the adults. In our mind, two facts suggest that neonate larvae may also participate in the selection of host fruit. Firstly, codling moth females lay a vast majority of eggs on foliage, not fruit [17], and it is the newly hatched neonate larva that ultimately decides which fruit is to be infested. Secondly, neonate larvae of codling moth exhibit a high potential for dispersal in their pursuit for host fruit. In preliminary experiments we continuously observed 100 neonate larvae for 18 h after hatch. On average, they traversed a distance of 19.3 T 0.47 m (mean T SEM, N = 100), while not eating and relying on yolk reserves in their gut only. We think that host fruit selection may be learned by codling moth neonates, and that single, shortlasting, post-hatch exposure to gustatory stimuli, provided by foliage sampling, may play a key role in this process. To date, two kinds of single experience learning were clearly demonstrated in insects during food selection processes: learning through preference induction and learning through aversive conditioning [14]. Insects that learn through preference induction will tend to prefer the food source (and taste stimuli associated with that source) they have already experienced, whether or not the chosen food is most appropriate for development [14,18,19]. Food preference induction is regarded to be non-associative learning [14]. On the other hand, food aversion learning relies on developing a negative association between the taste of food and subsequent illness, caused by detrimental post-ingestive effects [20 – 22]. Food associated with negative experience will be rejected. As well in vertebrates [20] as in insects [21,22] this kind of learning produces long-lasting behavioral effects. In our paper we will show that indeed, codling moth neonates learn how to choose host fruit through feeding preference induction and food aversion conditioning.

2. Materials and methods 2.1. Insects, foliage, fruit, chemicals, and feeding stations Infested apples were harvested from unmanaged orchards in June/July 2001 and 2002 in areas near Pullman, WA.

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Codling moths (C. pomonella L.) that emerged from these apples were held at 25 -C, 70% –80% r.h., under a L16 : D8 light– dark regime, and given saturated sucrose solution. These moths were allowed to oviposit on waxed paper. Neonates were collected 0.5– 1 h post-hatch and used for experiments. The circadian hatch began approximately 6 h into the photophase. Test foliage and uninfested fruit (Golden Delicious) were harvested in July/August 2001 and 2002 from the same unmanaged orchards near Pullman, WA. Leaves or apples were placed in zip-lock bags containing a cotton wick moistened with distilled water to prevent desiccation, transported to laboratory, and used for experiments within 1 h after harvest. Saccharin hemicalcium salt and Triton -100 (wetting agent), purchased from Sigma (St. Louis, MO, USA), were dissolved in double distilled water. Foliage or fruit was treated with 1% saccharin hemicalcium salt in 0.02% Triton -100 (hereafter referred as to saccharine), or 0.02% Triton -100 only (hereafter referred as to solvent). It was recently shown that codling moth neonates do sense saccharine at this concentration, and increase apple foliage consumption in presence of this chemical [23]. Neonates were exposed to foliage in feeding stations made of sandwich-like combination of reinforcement rings, masking tape and microscope slides. Each feeding station contained a 6 mm diameter circular section of leaf that neonates could individually feed on. Feeding intensity was determined by calculating the surface area of leaf consumed, and converting this surface to mass ingested. Details of arranging the feeding stations and evaluating leaf mass consumed are explained elsewhere [24]. 2.2. Preference induction Preference to saccharine-treated apple was induced by individually exposing the neonates to saccharine-treated apple foliage in feeding stations for the period of 3 h. Control group was exposed, in the same manner, to solventtreated foliage. After exposure, larvae were immediately subjected to double-choice assay between saccharine-treated apple and solvent-treated apple. Preparation of the choice assays are described in details in the paragraph ‘‘Choice assays’’. 2.3. Retention of preference induction Newly hatched neonates were individually exposed in feeding stations to saccharine-treated apple foliage, and allowed to feed for 3 h. Next, they were transferred to feeding stations containing untreated apple foliage and fed there for 1, 2, 3, 4, 6 or 21 h. Afterward, these larvae were subjected to double-choice assays between saccharine- and solvent-treated apple (see ‘‘Choice assays’’). In one experimental variant, induced larvae were subjected to host-choice assays immediately after exposure to saccharine. Control

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neonate larvae were subjected to the same procedures, except solvent was used instead of saccharine. For each retention time, 105 experimental and 105 control larvae were used, in 7 groups, 15 larvae each. The same experimental protocol was employed for another group of neonates, only these larvae were exposed to saccharine (experimental insects) or to solvent only (control insects) for 18 h after hatch. 2.4. Averse conditioning Averse conditioning to saccharine taste was done by concurrent exposure to saccharine and foliage of maidenhair tree, Gingko biloba, which was used as negative reinforcement. First, deleterious properties of Gingko leaves were investigated. Thirty-three neonate larvae were individually placed in feeding stations, on sections of Gingko leaves. Consumed amounts of leaves and mortality were recorded throughout 24 h, in 3-h intervals, as described elsewhere [24,25]. Another 33 neonate larvae were exposed to apple foliage in feeding stations and constituted a control group in this experiment, and these were monitored in the same manner as larvae fed Gingko leaves. This experiment delineated the amount of Gingko leaf tissue that needed to be ingested by larvae in order to provide non-lethal, but deleterious effects. Consumption of 7 –8 Ag (dry mass) of untreated Gingko leaf tissue did not cause mortality, but had definite emetic effects on the larvae. Consumption of 7 Ag dry mass of Gingko leaf was used as a negative reinforcement in following experiments. One hundred neonate larvae were individually exposed in feeding stations to Gingko leaves treated with saccharine and monitored for 3 h, in 15-min intervals. Upon consumption of 7 Ag dry mass of leaf, the larvae were transferred to feeding stations containing untreated apple foliage, allowed to individually feed there for subsequent 12 h, and finally subjected to choice assays described in section ‘‘Choice assays’’. Control group (N = 100) was treated in the same manner, except Gingko leaves were not treated with saccharine, but with solvent only.

2.6. Choice assays Uninfested, unripe apples, ca. 3 cm diameter were used for choice assays. Apples were submerged in 1% solution of saccharine in 0.02% aqueous Triton -100 or 0.02% aqueous Triton -100 (solvent) only, and allowed to air dry. A pair of apples (one saccharine-treated and one solvent-treated) was placed in 70 mm diameter Pyrex glass crystallizing dish, so the apples would touch the wall of the dish, and a distance ca. 1 cm between the fruits would be left. The leaf section with test larva feeding on it was removed from respective feeding station, gently placed in the middle of the distance in between the fruits and the crystallizing dish was covered by a glass Petri dish. To prevent airflow that could bias the results of the assay, the entire assembly was placed in semi-translucent 473 ml highdensity polypropylene container and covered with a transparent lid. The testing bench and taste arenas were illuminated by fluorescent tubes and positioned so luminosity (460 –480 lx) was constant for all tests. Glassware and polypropylene containers were washed in tap water, double distilled water, alcohol, acetone, and ethyl ether, and dried. Glassware was baked in 240 -C, overnight. Each assay lasted for 24 h under photoperiod synchronized to the stock culture. Larval behavior was observed during the period of time preceding apple infestation. Additionally, all apples were examined under dissecting microscope for evidence of burrowing. Once burrowing was found, the fruit was fragmented and infestation verified by identifying the larva. 2.7. Statistical analysis Exact Fisher test was used for evaluation of host fruit preference in choice assays. Retention data sets were subjected to ANOVA followed by multiple Bonferroni comparison. Student’s t-test was used to evaluate effects of Gingko leaves on feeding. Differences were regarded to be statistically significant at P < 0.05.

2.5. Retention of averse conditioning

3. Results

To evaluate retention of averse conditioning newly hatched neonate larvae were individually exposed in feeding stations of Gingko leaves treated with saccharine in a manner described in the previous paragraph. Next, the larvae were transferred to feeding stations containing untreated apple foliage, and allowed to individually feed there for a retention period: 1, 2 or 3 days, or until they molted to the next larval instar. Afterward, the larvae were subjected to choice assays between saccharine-treated apple and solvent-treated apple. Control neonate larvae were subjected to the same procedure, except the Gingko leaves were treated with solvent only. For each retention time, 105 experimental and 105 control larvae were used, in 7 groups, 15 larvae each.

3.1. Effects of post-hatch exposure to saccharine A 3-h exposure to saccharine elicited a significant preference for saccharine-treated apple. Neonates that had been exposed to saccharine during the training session exhibited two-fold behavioral pattern of fruit searching strategy. Fifty neonates (out of 99 tested) encountered a saccharine-treated apple as the first choice. All of them accepted the first host and burrowed into saccharine-treated fruit. Forty-nine larvae out of 99 tested neonates encountered a solvent-treated apple first, and continued searching for fruit, alternatively visiting each of presented apples at least once. Forty of these larvae burrowed to saccharinetreated apple upon the first visit, 4 upon the second visit,

M.A. Pszczolkowski, J.J. Brown / Physiology & Behavior 86 (2005) 168 – 175 Table 1 Host fruit preference induced with saccharine in codling moth neonates Variant

Experimental Control

Preference inductiona

3.2. Effects of post-hatch exposure to Gingko biloba leaves without saccharine

Choice of the host fruit (number of larvae infesting)

Saccharineb Solvent onlyc

Saccharineb

Solvent onlyc

94 49

5d 50

No mortality was observed in larvae fed apple foliage. Among larvae fed Gingko leaves, mortality (3%) was detected after 9 h of feeding, and reached approximately 80% by the end of exposure (24 h of feeding). No mortality was observed in larvae exposed to Gingko for 3 h, however all larvae regurgitated indicating a deleterious effect of feeding on Gingko. This finding was further supported by feeding intensity data. Throughout 3-h exposure, neonate larvae fed apple leaves consumed 22.3 T 0.21 Ag of dry mass (average T SEM, N = 33), whereas those exposed to Gingko leaves consumed significantly less (6.7 T 0.05 Ag average T SEM of dry mass, N = 33, P < 0.001, Student’s t-test).

a Continuous, 3-h exposure on apple foliage treated with test solution. Larvae were allowed to feed ad libitum. b 1% Saccharine in aqueous 0.02% Triton -100. c Aqueous 0.02% Triton -100. d P < 0.001, Fisher exact test.

Preference (% of population)

and 5 visited saccharine-treated fruit once, then abandoned it, visited the solvent-treated fruit and burrowed into it. In the control experiment, neonates that had not been exposed to saccharine prior to choice assays, did not discriminate between saccharine- or solvent-treated apples. All larvae tested (N = 99) visited each apple at least two times before randomly burrowing into one, and 62 of them needed three visits to make a choice of host fruit. Larval strategy of host searching was strictly mirrored by the pattern of fruit infestation. The vast majority (96%) of the neonates that had been exposed to saccharine, infested saccharine-treated apples (Table 1, Fisher exact test, P < 0.001). However, those that had not experienced saccharine before being subjected to choice assay, infested the fruit randomly (Table 1, Fisher exact test, P > 0.001). Preference of saccharine-treated apple was maintained for about 3 h after the training session had ended. Larvae without stimuli from saccharine for 4, 5, 6 or 21 h no longer preferred saccharine-treated over Triton -100-treated apples in double-choice assays (Fig. 1A, ANOVA P < 0.01). Similar results were obtained for the larvae that were conditioned by 18-h exposure to saccharine (Fig. 1B, ANOVA P < 0.01).

100

80

3.3. Effects of concurrent post-hatch exposure to Gingko biloba leaves and saccharine Concurrent exposure to saccharine and Gingko leaves had deleterious effects on codling moth larvae. All neonates regurgitated upon consumption of 7 Ag of foliage dry mass. We assume that these neonates associated the unpleasant experience caused by Gingko with the taste of saccharine. Larval strategy of fruit searching differed in control and experimental larvae. Those neonates that experienced concurrent exposure to saccharine and deleterious Gingko leaves exhibited two kinds of fruit searching behavior. Forty-nine out of 99 tested larvae encountered saccharinetreated fruit first, but none of them attempted to burrow. Instead, they abandoned that fruit, continued searching, and burrowed into solvent-treated apple upon the first visit. Fifty neonates from this group encountered solvent-treated apple first. Instead of burrowing into it, they continued searching for fruit, visited saccharine-treated apple once, and abandoned it with no attempts of burrowing into it. Next, they all

A

***

B

***

60

40 0

2

4

6

16

18

20

22

0

2

4

100

80

*** *** **

*** *** **

60

171

6

16

18

20

40 22

Hours after training Fig. 1. Retention of host preference induction in neonates of the codling moth. Newly hatched neonates were allowed to feed on apple leaves treated with 1% saccharine in 0.02% aqueous Triton -100 for 3 h (A, solid circles) or for 18 h (B, solid triangles), or to 0.02% aqueous Triton -100 alone for 3 h (A, open circles), or 18 h (B, open triangles). Conditioned larvae were transferred to untreated apple foliage for respective period of time (abscissae), and finally given a choice between saccharine- and Triton-treated apple. Retention is shown as mean percent of experimental population that responded to post-hatch treatment by infesting the saccharine-treated apple. Each datum point shows mean T SEM of 7 groups, 15 larvae each. ***P < 0.001, **P < 0.01, ANOVA followed by multiple Bonferroni comparison.

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Table 2 Averse conditioning of host fruit selection in codling moth neonates Averse conditioninga

Variant

Post-conditioning exposureb

Saccharinec, Gingko leaf Solvent onlyd, Gingko leaf

Experimental Control

Apple leaf, untreated Apple leaf, untreated

Choice of the host fruit (number of larvae infesting) Saccharinec

Solvent onlyd

5e 50

94 49

Exposure to Gingko leaves was used as negative reinforcement of saccharine taste. a Larvae were exposed to Gingko leaf and allowed to feed. Conditioning was terminated upon ingestion of 7 `ıg of dry mass. b Larvae were exposed to untreated apple leaf and allowed to feed ad libitum for 12 h following the conditioning. c 1% Saccharine in aqueous 0.02% Triton -100. d Aqueous 0.02% Triton -100. e P < 0.001, Fisher exact test.

Avoidance (% of population)

visited the solvent-treated apple again and burrowed into it. In control experiment, neonates that had not been exposed to saccharine and Gingko prior to choice assays did not discriminate between saccharine- or solvent-treated apples. All larvae tested (N = 99) visited each apple at least two times before randomly burrowing into the fruit. Sixty of them needed three visits to choose a suitable host. The pattern of fruit infestation strictly mirrored the behavior of the neonates. Only 5 out of 99 tested larvae infested saccharine-treated fruit in host fruit choice assays (Table 2, Fisher exact test, P < 0.001). None of saccharinetreated apples, except those five that were infested, showed any traces of attempted penetration through the peel. The conditioned aversion to saccharine-treated apple was maintained for more than 3 days (Fig. 2), and could be observed even after molting to the second instar, i.e. 4 or 5 days after training, (Fig. 2B). Even then, more than 70% of larvae conditioned by concurrent exposure to Gingko and saccharine avoided infestation of saccharine-treated fruit.

100

80

A

B

*** *** *** ***

60

40 1

2

3

1

Day of instar Fig. 2. Retention of adverse conditioned host avoidance behavior in neonates of the codling moth. Newly hatched neonates were allowed to feed for 3 h on Gingko biloba leaves treated with 1% saccharine in 0.02% aqueous Triton -100 (solid circles), or with 0.02% aqueous Triton -100 alone (open circles). Conditioned larvae were transferred to untreated apple foliage for respective period of time (abscissae), and finally given a choice between saccharine- and Triton-treated apple. Retention is shown as mean percent of experimental population that responded to post-hatch treatment by avoiding saccharine-treated apple. A— first instar larvae, B— larvae after molting to their second instar. Each datum point shows mean T SEM of 7 replications, 15 larvae each. ***P < 0.001, **P < 0.01, ANOVA followed by multiple Bonferroni comparison.

4. Discussion There is an increasing body of evidence that host fruit odor (kairomones) attract codling moth neonates from a distance. These are a-farnesenes, mostly (E,E)-a-farnesene, [26,27], hexyl hexanoate [28] and methyl (2E, 4Z)-2,4decadienoate [29]. There is also an anecdotic evidence that (E,E)-a-farnesene liberates biting and burrowing behaviors in codling moth neonates [30]. However, the attracting action of fruit odors cannot solely lead to successful host infestation. First, (E,E)-a-farnesene itself does not arrest codling moth neonates that have arrived at the apple, and does not prevent further exploring of micro-environment by these larvae [30]. Secondly, literature shows that aforementioned kairomones are produced by various sources, many of them being unsuitable as hosts to codling moth neonates. They are produced by cotton [31,32], olives [33], citrus fruits [34,35], avocado [36], maize [37], oilseed rape flowers [38], kiwi [39], or passion fruit [40], to name only a few plant sources. They also are components of some insect pheromones [41 –44]. Consequently, codling moth neonates may be attracted to a kairomone source that would not support their development. We think that kairomones are of minor importance in ultimate selection of host fruit by codling moth neonates. Instead, we emphasize the role of post-hatch single experience learning in host selection by codling moth larvae. In the light of our previous and present results it seems that newly hatched codling moth neonates probe their micro-environment in search for chemical stimuli [12,23 – 25], and on the basis of this experience subsequently chooses suitable host fruit (this study). If stimuli induce and/ or increase feeding, or do not produce deleterious consequences upon ingestion, then the search for the host fruit is continued, and the larva chooses the fruit that has chemical characteristics resembling previously sensed taste. However, if the post-hatch exposure produces a deleterious effect (in our study — regurgitation, decreased food consumption and intoxication) an association is made between that stimuli and the negative experience. The search for the host fruit is continued, but here the neonate avoids hosts that chemically resemble a previously sensed taste associated with intoxication. In both cases exposure to

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the taste of saccharine does not need to last long to form an experience; 3-h exposure is sufficient to re-program the choice of the host fruit (Tables 1 and 2). These two kinds of experiences fall into two categories of learning involved in host selection by phytophagous insects: induction of preference, and food aversion learning. Codling moth neonate larvae were exposed to saccharine only in a manner that is typically used for evoking food induction preference [14,18]. There was no association provided in between the taste of saccharine and any kind of reinforcement, thus we assume that here the neonates were subjected to non-associative learning. Induction of feeding preference is commonly found in lepidopteran larvae. In his review [19], Jermy mentions some form of food preference induction in 24 lepidopteran species, ranging from strictly monophagous Papilio mahaon [45] to Hyphantria cunea that feeds on over 200 unrelated plant species [46]. However, our current work extends the general knowledge about lepidopteran larvae’s capability of learning by preference induction. Careful analysis of the references in Jermy’s review shows that induction of food preference was found in neonates of two species only; Helicoverpa zea [47], and Manduca sexta [48]. Also, only one reference [49] demonstrated that acquisition time for preference induction may be as short as that found in our study. Retention of preference induction found in our experiments is surprisingly short-lasting in comparison to data published for other species (see Ref. [19]): in the codling moth it was retained only for 3 h, regardless of the length of conditioning to the inducing factor (Fig. 1). Generally, feeding preference, once induced in lepidopteran larvae, may persist through one or several weeks [18,49,50]. The adaptive advantage of long-lasting food preference induction has been assumed to reduce the probability that in a mixed plant stand an insect would frequently and repeatedly change food species. This type of learning is reinforced by natural selection since frequent and repeated changing of food source decreases efficiency of food utilization [19]. Frequent and repeated changing of host fruit is not the case for codling moth neonates thus long-lasting host preference induction would have minor adaptive value here. However, if frequent and repeated visiting of various potential host fruit is codling moth neonates’ behavior, then rapidly formed and short-lasting food preference induction may have some adaptive value. Codling moth’s great potential to challenge new host plants raises a question of what role in regulation of host selection behavior could be attributed to neonates’ feeding on leaves before entering the ultimate host — the fruit. Some of our previously published data show that just after hatch codling moth neonates use a simple form of learning to modify their feeding behavior [25]. On the other hand, it has been shown that codling moth possesses a genome which can be shifted to produce adaptive responses that develop stable trophic relationships with tree species other than apple [7]. If we assume that codling moth evolved in a

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dense, primeval forest that included intertwining canopies of primitive apple (the predominant host of codling moth) and other trees, then it is reasonable to assume that codling moth neonates challenged multiple host trees while searching for a suitable food source. Theoretically, codling moth larvae may have been solely foliage feeders similar to their leafroller relatives today (Tortricidae), and they probably sampled small individual or clustered fruit when these were encountered in order to satisfy their dietary need for protein. If so, natural selection pressure probably favored those larvae that shifted feeding behavior toward entering larger fruit, thereby utilizing a protected location from predators, less danger of dehydration or fire, ample amounts of simple carbohydrates, and protein rich seeds. Consequently, would it not be surprising that codling moth still exhibits some behaviors that, at early stages of its phylogenesis, helped distinguish suitable from unsuitable host fruit? Perhaps, leaf feeding helps neonate larvae avoid inappropriate, and reinforces appropriate host fruit selection? If so, even if none of the fruits available within the searching distance could be identified as an acceptable host, then after several hours the neonate loses the preference induction experience, and may start re-learning and searching again within the pool of available hosts. We will further elaborate on advantage of short-time learning in the final paragraph of this discussion. The neonates concurrently exposed to saccharine and deleterious Gingko foliage during the training session associated taste of saccharine and negative experience caused by Gingko, and consequently avoided the taste of saccharine. This is a typical response known as food aversion [20]. Food aversion learning had a much longer lasting consequence in our experiments; aversion, once learned, persisted for longer than three consecutive days, and was not significantly disrupted even after molting to the next instar (Fig. 2). Long retention time of a negative experience reinforced with deleterious food is characteristic for food aversion learning in rats [51]. Data shown here on food aversion learning in the codling moth indicate that aversive conditioning in insects is robust too. An extensive discussion regarding the dynamics of food aversion learning in codling moth is difficult, because there are only a few references to this phenomenon in insects. Only one paper suggests such a form of learning exists in caterpillars [21], and one other paper [22] clearly demonstrates insect taste aversion conditioned as in our study, i.e. according to conditioning paradigm widely used in vertebrates [20]. The adaptive advantage of food aversion learning is self-evident: it prevents the consumption of deleterious or poisonous food. It was suggested [21,22,52] that mono- and oligophagous herbivores should not demonstrate such a type of learning, whereas polyphagous ones should. The occurrence of conditioned food aversion in codling moth neonates supports previous postulates that young larvae of this species are polyphagous [6 –8,10,12].

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To our knowledge there has been only one report [50] that showed that feeding preference may be induced as rapidly as we have demonstrated in codling moth. We are not aware of any reports that feeding preference in insects can be forgotten so quickly. Our finding that codling moth neonate can learn and forget quickly is worth emphasizing because previously reported rigidity of induced food preference puzzled entomologists. There are at least three papers showing that lepidopteran larvae induced by feeding on one host plant, and transferred to another host plant (that normally would be consumed) do not feed at all and finally die [49,53,54]. This phenomenon analyzed and named by Jermy [19] as ‘‘starving-to-death-at-Lucullian-banquets’’ has clearly no adaptive value and may even reduce fitness in certain ecological situations. It also was considered to be evidence of limited flexibility of caterpillars’ primitive nervous system. Surprisingly, it is not the case of apparently naı¨ve codling moth neonates that increase their fitness by developing two mutually complementing mechanisms of host fruit selection, both having adaptive significances. To the best of our knowledge, such a combination has not been reported before.

Acknowledgements This work was partially supported by Washington State Tree Fruit Research Commission, Washington State Commission on Pesticide Registration, and IFAFS and RAMP funding from the U.S. Department of Agriculture.

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