| Chapter 5. The role of honeydew in host searching of aphid hyperparasitoids | ||
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| Chapter 5. The role of honeydew in host searching of aphid hyperparasitoids | ||
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Table des matières
Foraging in many insect parasitoids is mediated by chemicals associated with their hosts. For example, honeydew, the faeces of feeding aphids induces and/or prolongs the searching behaviour of aphid parasitoids. In the laboratory, we tested if aphid hyperparasitoids, which belong to a higher trophic level, also rely on aphid honeydew to locate their hosts. To do this we used the potato aphid, Macrosiphum euphorbiae, the primary parasitoid, Aphidius nigripes and four hyperparasitoids, Asaphes suspensu s, Dendrocerus carpenteri, Alloxysta victrix , and Syrphophagus aphidivorus that possess different biological attributes and host ranges. In addition we determined if foraging hyperparasitoid females could discriminate between (i) honeydew from a host and a non-host (the potato aphid and the soft brown scale, Coccus hesperidum ), and (ii) honeydew from healthy aphids and those parasitised by A. nigripes . Females of A. suspensus did not react to any of the honeydew treatments. While the presence of non-aphid honeydew did not modify the behaviour of A. victrix , D. carpenteri and S. aphidivorus females, they exhibited a significant increase in searching time and path length, but not walking speed when in the presence of honeydew from aphids. However, there were no changes in host searching behaviours, such as antennation or ovipositor probing, that have been reported for primary aphid parasitoids. There was no significant difference in the response of hyperparasitoid females to honeydew from healthy and parasitised aphids. These results indicate that hyperparasitoids may use aphid honeydew, a conspicuous cue from the second trophic level, as an infochemical to locate their hosts.
Key Words - Honeydew, aphid, aphid parasitoid, hyperparasitoid, host searching behaviour, trophic interactions, infochemical detour.
Chez plusieurs parasitoïdes d’insectes, la recherche de l’hôte est modulée par des infochimiques associés à l’hôte. Par exemple, le miellat, ou l’excrétion fécale des pucerons, induit ou prolonge le comportement de recherche chez les parasitoïdes de pucerons.
Au laboratoire, nous avons determiné si les hyperparasitoïdes de puceron, qui appartiennent à un niveau trophique supérieur, utilisent également le miellat pour localiser leurs hôtes. Nous avons utilisé le puceron de la pomme de terre, Macrosiphum euphorbiae , le parasitoïde, Aphidius nigripes et quatre hyperparasitoïdes, Dendrocerus carpenteri , Asaphes suspensus , Alloxysta victrix et Syrphophagus aphidivorus . Nous avons déterminé si les femelles hyperparasitoïdes avaient la capacité de discriminer entre (1) le miellat excrété par le puceron M. euphorbiae et celui de la cochenille, Coccus hesperidum , laquelle n’abrite pas d’hôtes potentiels, et (2) le miellat de pucerons sains et celui des pucerons parasités par A. nigripes .
Les femelles A. suspensus n’ont répondu à aucun des traitements de miellat, alors qu’aucun hyperparasotoïde n’a répondu au miellat de cochenille. Au contraire en présence du miellat de puceron, le temps de recherche et la longueur des tracés, mais pas la vitesse de marche, ont augmenté chez A. victrix , D. carpenteri et S. aphidivorus . Toutefois, leurs femelles n’ont pas réagi au miellat par certains comportements spécifiques observés chez les parasitoïdes primaires de pucerons, tel l’investigation avec les antennes ou l’ovipositeur. De plus, les femelles hyperparasitoïdes n’ont pas discriminé entre le miellat de pucerons parasités et non-parasités.
Ces résultats montrent que les hyperparasitoïdes pourraient utiliser le miellat de puceron, une substance manifeste du deuxième niveau trophique, comme infochimique pour localiser leurs hôtes.
Honeydew, a complex mixture of chemical compounds, of which the most important are sugars and amino acids (Auclair, 1963) is excreted by phloem-feeding Homoptera, such as aphids, whiteflies and scale insects. Differences in chemical composition of aphid honeydew have been studied in detail and may vary depending on the host plant species (Hendrix et al. , 1992; Douglas, 1993; Fisher and Shingleton, 2001), the aphid species (Hendrix et al. , 1992; Völkl et al. , 1999; Fisher and Shingleton, 2001), the aphid age (Fisher et al. , 2002), the sugar concentration in the diet (Mittler and Meikle, 1991; Wilkinson et al. , 1997), the level of ant tending (Fisher and Shingleton, 2001; Yao and Akimoto), the presence of bacterial intracellular symbionts (Sasaki et al. , 1990; Wilkinson and Douglas, 1995; Wilkinson et al. , 1997), and parasitism (Cloutier 1986). Honeydew may serve as a source of carbohydrates for many insects, for example ants and parasitoids (Völkl et al. 1999; Wäckers and Steppuhn, 2003).
Honeydew is also used as an infochemical by foraging parasitoids (e.g. Bouchard and Cloutier 1984) and predators (e.g. Budenberg and Powell, 1992). Its role in host searching of aphid parasitoid females has been studied extensively. Honeydew attracts foraging parasitoid females (Wickremasinghe and van Emden, 1992; Bouchard and Cloutier, 1985) and/or arrests them on contaminated areas (Bouchard and Cloutier, 1984; Gardner and Dixon, 1985; Ayal, 1987; Budenberg, 1990; Cloutier and Bauduin, 1990; Hågvar and Hofsvang, 1991; Budenberg et al. , 1992; Grasswitz and Paine, 1993). Honeydew may also contain substantial specific information for natural enemies, for while Aphidius rhopalosiphi females respond to honeydew of both host and non-host aphids, they spend less time in areas contaminated with honeydew from the non-host species (Budenberg, 1990).
Aphid parasitoids can in turn be parasitised by different species of hyperparasitoids. Contrary to primary parasitoids, honeydew from healthy aphids does not appear to attract hyperparasitoids towards contaminated areas (Buitenhuis et al. , unpublished). This is not altogether surprising for while honeydew is a direct cue to the presence of aphids for parasitoids, it would only be an indirect cue for hyperparasitoids as it provides females no reliable information about the presence of their aphid parasitoid hosts. On the other hand, honeydew does act as a contact synomone, inducing hyperparasitoid females to stay and search longer on contaminated surfaces and plants (Budenberg, 1990; Grasswitz, 1998; Buitenhuis et al. , unpublished). However, parasitism by braconid wasps may also induce changes in both the quantity and composition of honeydew produced by aphids (Cloutier and Mackauer 1979, Cloutier 1986, Rahbé et al. , 2002). Therefore, honeydew could be a direct and reliable cue for hyperparasitoids if females have evolved the capacity to discriminate between honeydew from healthy and parasitised aphids.
In this study, we examined the innate response of aphid hyperparasitoids to different types of honeydew. We predicted that foraging hyperparasitoid females not only have the ability to detect honeydew but also show a preference for honeydew from aphid rather than non-aphid species and, more specifically, for honeydew from parasitised aphids. We tested these predictions in the laboratory by measuring behavioural components of hyperparasitoid females exposed to water extract of honeydew applied to filter paper discs following the study of Bouchard and Cloutier (1984). We used the potato aphid, Macrosiphum euphorbiae (Thomas) , its primary parasitoid, Aphidius nigrip es Ashmead and four hyperparasitoids, Asaphes suspensu s Walker (Pteromalidae), Dendrocerus carpenteri (Curtis) (Megaspilidae), Alloxysta victrix (Westwood) (Alloxystidae), and Syrphophagus aphidivorus (Mayr) (Encyrtidae). These species were chosen for while they all naturally exploit Aphidius spp. they possess different biological attributes and host ranges. A. suspensus and D. carpenteri are generalist ectoparasitoids which attack primary parasitoid prepupae or pupae following mummification of the aphid. A. victrix is an endoparasitoid that lays its egg in parasitoid larvae prior to aphid mummification and has a more restricted host range. Finally, S. aphidivorus is a generalist hyperparasitoid with the capacity to attack either primary parasitoid larvae in live aphids or parasitoid prepupae or pupae following mummification.
Colonies of the aphid, parasitoid and four hyperparasitoids were reared on potato seedlings following the techniques of Brodeur and McNeil (1994) and Buitenhuis et al. (submitted).
To prevent contact with honeydew stimuli before the test, hyperparasitised aphid mummies were collected, put in individual gelatine capsules and kept at 20±1ºC, 75±10% RH, under a 16L:8D photoperiod until adult emergence. The adults were then sexed and females were put in small individual ventilated cylindrical cages (5 cm in diameter and 10 cm in height) with a supply of 40% sucrose solution and held under the same conditions until used in the experiment.
Aphid honeydew was collected by placing Parafilm™ sheets for 24 h under potato plants infested with either healthy aphids from all developmental stages or parasitised aphids. Parasitised aphids were obtained by exposing third instar aphids for 24 hr to 3-5 day-old mated females at a parasitoid: host ratio of 1:2 (resulting in 90-95% parasitism). Honeydew was collected 4-7 days later. The response of hyperparasitoid females to honeydew from a non-host was tested using honeydew from scale insects ( Coccus hesperidum L.: Coccidae) collected on Ficus benjamina L. (Moraceae) plants. In this instance, both the herbivore and the plant species were different from the potato-aphid system. This honeydew was collected in the same manner as described above but, due to the lower insect density, the Parafilm sheets were removed after 2 days. Honeydew was allowed to dry for 30 minutes at 40°C, collected by scraping the sheets with a glass microscope slide and then stored at -20°C until use. Before the bioassay, the honeydew was weighed and dissolved in distilled water, filtered through a cloth, and adjusted to a concentration of 0.26 mg/µl (following Bouchard and Cloutier, 1984). Between bioassays the solution was stored at 4°C for a maximum of 8 days.
100 µl of the distilled water, honeydew from either healthy or parasitised aphids, or from non-host scales was applied in the middle of a filter paper (12.5 cm diameter; Schleicher & Schuell #595), giving a treated circle about 4 cm in diameter. A circle of 12 cm in diameter was drawn inside the perimeter of the disc. The paper was dried under laboratory conditions and used within 5 hours of preparation. For each assay the test paper was placed in a 14 cm diameter glass petri dish, covered by a glass plate, located in a tent lit by a circular 22W fluorescent tube. One virgin, naïve, female hyperparasitoid (2-7 day-old) was released onto the middle of the filter paper and her behaviour recorded on video until she either crossed the 12 cm circle or until she flew to the side or top of the arena. Females that immediately flew off the filter paper or that did not move were excluded from the analysis. One female of each of the four hyperparasitoid species was tested on the same filter paper. The filter papers treated with aphid honeydew were only used once, but in the case of scale honeydew assays they were used twice due to the shortage of scale honeydew solution. Such a procedure had no effect on any of the measured parameters: residence time, path length, walking speed (2-way ANOVAs using hyperparasitoid species and 1st/2nd repetition as factors, all p-values >0.05). In all assays the hyperparasitoid species were randomised within the treatments, and 20 replicates per treatment were done within a 8 day period.
The time spent inside and outside the contaminated area was determined from the videotape using the Observer© (Noldus, 1997, version 3 for Macintosh), while the locomotory behaviour was quantified by tracing each female’s path on a transparency and then measuring its length. Walking speed (cm/s) was calculated by dividing the total path length by the total time.
Duration and path length data were log(x+1) transformed, whereas speed data were square root transformed prior to be analysed using a two-way ANOVA. Differences between treatments were determined by contrasts. Given that statistical models had 3 degrees of freedom per factor, only 3 orthogonal contrasts (α=0.05) were allowed. To test the predictions, we selected a priori (i) honeydew, regardless of origin vs water, (ii) aphid vs non-aphid (scale) honeydew, and (iii) honeydew from healthy and parasitised aphids. One additional contrast analysis, (iv) non-aphid (scale) honeydew vs water, was done using Scheffé’s adjustment of the p-value (Steel and Torrie, 1980). Differences between hyperparasitoid species were determined by Fisher’s protected LSD (α=0.05). All analyses were done using SAS (SAS Institute, 1999).
Most females of all hyperparasitoid species walked on the filter paper: A. victrix (77%), A. suspensus (88%) D. carpenteri (94%), and S. aphidivorus (100%). In all species, walking was continuous or could be interrupted with short jumps. The trajectories of females that did or did not respond to honeydew were very different. Females in the control treatment, and those not responding to honeydew, usually walked rapidly across the treated area without showing evidence of arrestment (Figure 5-1). In contrast, a positive response was characterised by a klinotactic response, and the resulting tortuous path ensured that the females searched most of the treated area (Figure 5-1).
Overall, there were significant effects of both treatment and species on residence times and path length (Table 5-1, Figures 5-2 and 5-3). However, while there were species specific differences in walking speed, for any given species, this parameter was unaffected by treatment (Table 5-1, Figure 5-4).
Clearly, these treatment effects are due to overall differences in response to aphid honeydew compared with those to water and honeydew from scale insects (Table 5-2, Figures 5-2 and 5-3). However, the contrast analyses underlined specific treatment differences between the four hyperparasitoid species (Table 5-2). One noticeable point is that A. suspensus female foraging behaviours remained unchanged in all assays (Table 5-2, Figures 5-2 and 5-3). The apparent increase in time spent in the scale insect treatment on Figure 5-2 was non-significant and resulted from the behaviour of two females, one which spent a long time walking in the treated patch and the other which remained outside the patch for a prolonged period. When the pooled responses to honeydew, regardless of source, and water were contrasted, D. carpenteri females showed significant changes in foraging while A. victrix and S. aphidivorus did not. However, there are no differences between water and scale honeydew for any given species (Table 5-2), while all respond to aphid honeydew.
Contrary to our initial hypothesis, there were no differences between honeydew from healthy and parasitised aphids (Table 5-2, Figures 5-2 and 5-3).
Our results, together with those of Budenberg (1990) on Alloxysta macrophadna and Phaenoglyphis villosa (Alloxystidae), and Grasswitz (1998) on A. victrix , indicate that aphid honeydew may modify female foraging behaviour in species from each of the three superfamilies (Cynipoidea, Ceraphronoidea, Chalcidoidea) where aphid hyperparasitoids are found. The existence of such a common response among evolutionary diverse groups of aphid hyperparasitoids would suggest that aphid honeydew is a reliable cue to host finding and may thus serve as a contact synomone that transcends trophic levels. A parallel study, at a different spatial scale using whole plants, also showed that the foraging behaviour of hyperparasitoid females was significantly modified by the presence of aphid honeydew (Buitenhuis et al. , unpublished). This conclusion is supported by the fact that the behavioural changes observed were not in response to all sources of honeydew, but rather to honeydew produced by insects serving as a host for the primary parasitoid. This ability to discriminate between aphid and non-aphid honeydew would result in females making extensive searches in areas where aphid parasitoids are most likely to be found. Honeydew composition is in a large part determined by the elements of phloem sap, and is thus partly plant specific (Hendrix et al. , 1992), so we cannot exclude the possibility that the different patterns we observed may be associated with the different host plants, i.e. potato vs Ficus plants used by the two herbivore species. However, discrimination between host and non-host honeydew has been reported, as the whitefly parasitoid Encarsia formosa responded differently to whitefly and aphid honeydew, when both species are reared on the same host plant (Romeis and Zebitz, 1997).
It is evident that not all hyperparasitoids respond in the same way to honeydew from hosts exploited by primary parasitoids, for while three of the four species modified their behaviour, one, A. suspensus , did not. Furthermore, no consistent patterns of response to aphid honeydew are found when considering aphid hyperparasitoids with different life-history strategies (endo- vs ectoparasitoids, koino- vs idiobiont parasitoids) and the stage of primary parasitoid host attacked (parasitoid larva in live aphid or parasitoid pupa in aphid mummy). Similarly, host specificity does not appear to shape aphid hyperparasitoid responses to honeydew. For example, A. victrix , a koinobiont hyperparasitoid, has a narrower host spectrum than most idiobiont hyperparasitoids, including those tested in this study (Brodeur 2000), but showed the same type of response. In contrast, the foraging of A. suspensus , a cosmopolitan and polyphagous species (Höller et al . 1993), was unaffected by honeydew on the substrate (this study). However, it was arrested by aphids/honeydew on plants (Buitenhuis et al. , unpublished). Nevertheless, it is possible that the use of honeydew as a foraging cue can be learned. Clearly more species must be examined in order to explain such marked differences in preference or absence of response.
Despite the potential advantages of recognising honeydew from parasitised aphids, females of the hyperparasitoid species we tested did not discriminate between honeydew from healthy and parasitised aphids. Several non-exclusive explanations may account for this. First, differences between honeydew from healthy and parasitised aphids are mostly reflected in quantitative differences in the concentrations of amino acids being measured (Cloutier, 1986). Furthermore, while the presence of primary parasitoid larvae may modify aphid honeydew, several other factors may result in similar changes. These include aphid and host plant species, which may modify the nature and concentration of amino acids and sugars present (Douglas, 1993; Völkl et al. , 1999; Fisher and Shingleton, 2001). In addition, hyperparasitoid females foraging in an aphid colony under natural conditions will encounter a mix of new and decomposing honeydews from both healthy and parasitised aphids, which could mask any subtle quantitative differences associated with the origin of the synomone. One must therefore conclude that differences between honeydew from healthy and parasitised aphids do not provide sufficiently reliable cues to modify foraging behaviour.
Primary parasitoids and hyperparasitoids of aphids both use aphid honeydew in host searching and the response to this cue appears to be innate as naïve females respond to the infochemical (Bouchard and Cloutier, 1984; Grasswitz 1998; Grasswitz and Paine, 1993; this study). There are, however, distinct differences between the two trophic levels. While both use honeydew as an arrestment cue, primary parasitoids use volatiles from honeydew in long distance search (Bouchard and Cloutier, 1985), while hyperparasitoids do not (Buitenhuis et al ., unpublished). Furthermore, when primary parasitoids contact host honeydew there are a series of behavioural changes, including increased antennation, abdominal extension/flexing, reduced walking speed and increased turning rate (Bouchard and Cloutier, 1984; Budenberg, 1990; Hågvar and Hofsvang, 1991). Female hyperparasitoids also spend longer times and follow tortuous paths when encountering honeydew patches, yet they maintain a constant walking speed and do not perform the specific behaviours seen in the primary parasitoids. Such differences may arise from differences in the reliability of aphid honeydew as a foraging cue for primary and secondary parasitoids. Honeydew comes from the aphid and represents a reliable, abundant, and direct source of information about the presence of hosts to primary parasitoids (Vet and Dicke, 1992). In contrast, aphid honeydew provides no reliable information about the availability of suitable stages of the primary parasitoid that the hyperparasitoid females exploit. This situation represents an original example of an « infochemical detour », where the cue is only indirectly related to its host/prey (Vet and Dicke, 1992). Aphid hyperparasitoid females could benefit from searching in habitats contaminated by honeydew, as parasitised aphids and aphid mummies can either be found within or near the aphid colony (Brodeur and McNeil, 1989, 1992; Müller et al ., 1997). Furthermore, by keeping a constant walking speed, females possibly cover a greater area and thus gain the greatest benefit from an indirect cue for host availability.
We thank M. Bisson for technical assistance, M. Fréchette for identification of the soft brown scale, and Drs. W. Völkl, A. Chow and T. Grasswitz for providing hyperparasitoids. This study was supported by grants from the Natural Sciences and Engineering Research Council of Canada to J.B. and J.N.M., as well by graduate scholarships from the Centre de Recherche en Horticulture and Agriculture and Agri-Food Canada to R.B.
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Table 5-1 Results of 2-way ANOVAs on different parameters describing the behaviour of four species of aphid hyperparasitoids foraging on a filter paper disc treated with different honeydew extracts.
|
Factors |
|||
|
Honeydew treatment |
Hyperparasitoid species |
Interaction |
|
|
Total residence time |
F3,252=11.78, P <0.001 |
F3,252=3.43, P=0.018 |
F9,252=1.40, P=0.186 |
|
In treated patch |
F3,252=8.62, P <0.001 |
F3,252=4.91, P=0.002 |
F9,252=1.79, P=0.071 |
|
Outside treated patch |
F3,252=9.16, P <0.001 |
F3,252=3.63, P=0.014 |
F9,252=0.56, P=0.827 |
|
Path length |
F3,252=16.62, P <0.001 |
F3,252=26.78, P <0.001 |
F9,252=1.03, P=0.413 |
|
Walking speed |
F3,252=1.53, P=0.207 |
F3,252=54.42, P <0.001 |
F9,252=0.51, P=0.868 |
Table 5-2 P-values of contrast analyses on the effect of different types of honeydew on the behaviour of four species of aphid hyperparasitoids. Contrast treatments were: Control (distilled water); All honeydew (combination of all honeydew treatments); Aphid (honeydew produced by Macrosiphum euphorbiae , both healthy and parasitised by Aphidius nigripes ); Scale insect (honeydew produced by Coccus hesperidum ); Healthy aphid (honeydew produced by M. euphorbiae ); Parasitised aphid (honeydew produced by M. euphorbiae parasitised by A. nigripes ). Significant contrasts are indicated by asterisks ( P <0.05)
|
Contrast |
Visit time |
Path length |
Walking speed |
|||
|
Inside honeydew |
Outside honeydew |
Total |
||||
|
Alloxysta. |
Control vs all honeydew |
0.386 |
0.133 |
0.187 |
0.252 |
0.879 |
|
victrix |
Scale insect vs aphid |
0.018* |
0.080 |
0.020* |
0.009* |
0.569 |
|
Healthy aphid vs parasitised aphid |
0.844 |
0.685 |
0.762 |
0.844 |
0.840 |
|
|
Control vs scale insect 1 |
0.410 |
0.988 |
0.641 |
0.437 |
0.650 |
|
|
Asaphes |
Control vs all honeydew |
0.127 |
0.240 |
0.177 |
0.130 |
0.571 |
|
suspensus |
Scale insect vs aphid |
0.178 |
0.494 |
0.722 |
0.418 |
0.090 |
|
Healthy aphid vs parasitised aphid |
0.656 |
0.943 |
0.774 |
0.409 |
0.544 |
|
|
Control vs scale insect 1 |
0.052 |
0.639 |
0.223 |
0.517 |
0.554 |
|
|
Dendrocerus |
Control vs all honeydew |
0.004* |
0.308 |
0.008* |
0.002* |
0.310 |
|
carpenteri |
Scale insect vs aphid |
0.002* |
0.001* |
<0.001* |
<0.001* |
0.547 |
|
Healthy aphid vs parasitised aphid |
0.098 |
0.595 |
0.386 |
0.452 |
0.389 |
|
|
Control vs scale insect 1 |
0.770 |
0.244 |
0.796 |
0.983 |
0.687 |
|
|
Syrphophagus |
Control vs all honeydew |
0.092 |
0.128 |
0.057 |
0.003* |
0.125 |
|
aphidivorus |
Scale insect vs aphid |
0.038* |
0.004* |
0.003* |
0.004* |
0.873 |
|
Healthy aphid vs parasitised aphid |
0.736 |
0.234 |
0.348 |
0.628 |
0.478 |
|
|
Control vs scale insect 1 |
0.948 |
0.571 |
0.755 |
0.558 |
0.204 |
|
1Additional contrast with Scheffé adjustment.
Figure 5-1 Typical path tracings of aphid hyperparasitoid females that responded (left, Dendrocerus carpenteri on honeydew from parasitised aphid) or not (right, D. carpenteri on honeydew from scale insect) to honeydew.
Figure 5-2 Residence time (mean + SE) of female of four species of aphid hyperparasitoids foraging on a filter paper disc (12 cm in diameter) treated with different honeydew extracts. The bars further indicate the time spent within (black) and outside (white) of of the treated area. Per species, significant contrasts are indicated with horizontal bars. For details on all statistical differences, see Table 2.
Figure 5-3 Path length (mean + SE) of female of four species of aphid hyperparasitoids foraging on a filter paper disc (12 cm in diameter) treated with different honeydew extracts. Per species, significant contrasts are indicated with horizontal bars. For details on all statistical differences, see Table 2.
Figure 5-4 Walking speed (mean + SE) of female of four species of aphid hyperparasitoids foraging on a filter paper disc (12 cm in diameter) treated with different honeydew extracts. No species showed significant differences between treatments.
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| Chapter 4. Foraging behaviour on the fourth trophic level: a comparative study of host location in aphid hyperparasitoids. |
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Chapter 6. Conclusion |