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An introduction device for the aphidophagous hoverfly Episyrphus balteatus (De Geer) (Diptera: Syrphidae)
Biological Control 54 (2010) 181–188
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An introduction device for the aphidophagous hoverfly Episyrphus balteatus (De Geer) (Diptera: Syrphidae) Pascal D. Leroy *, François J. Verheggen, Quentin Capella, Frédéric Francis, Eric Haubruge Department of Functional and Evolutionary Entomology, University of Liege, Gembloux Agro-Bio Tech, Passage des Déportés 2, B-5030 Gembloux, Belgium
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Article history: Received 16 November 2009 Accepted 10 May 2010 Available online 31 May 2010 Keywords: Biological control Augmentative biocontrol Aphid Acyrthosiphon pisum Aphis fabae Megoura viciae Myzus persicae Aphidophagous Beneficial Natural enemies Predator Syrphid Episyrphus balteatus Oviposition induction Semiochemicals E-(b)-farnesene R-(+)-limonene (Z)-3-hexenol Honeydew Artificial honeydew
a b s t r a c t Augmentative biocontrol constitutes a safe option to reduce pest populations through the enhancement of natural enemies’ activity. In this context, the aphidophagous syrphid Episyrphus baltetaus (De Geer) (Diptera: Syrphidae) is a promising candidate for aphid biological control: larvae of this syrphid attack and consume a wide range of aphid species and are found on many vegetable crops. Because natural populations of beneficial insects are not always sufficient to regulate the pest infestations, this work has focused on the conception of a biological control device containing syrphid eggs which ones can easily be introduced in fields or greenhouses. Using semiochemicals [E-(b)-farnesene, R-(+)-limonene and (Z)-3-hexenol], honeydews and ‘‘artificial honeydews” (10% or 30% aqueous solutions of sucrose, fructose and glucose), the syrphid oviposition was artificially induced on an inert surface. Specifically, E-(b)-farnesene and concentrated mono-sugars (30%) were identified as the most efficient ovipositional stimulants. To test and validate the biological control device described above, laboratory and field experiments were performed: a plastic lamella covered with syrphid eggs was suspended on aphid infested plants in order to measure the efficiency of the device. The results obtained were promising since populations of 500 aphids were eliminated in 10 days when 15 syrphid eggs were introduced. The use of such a biological control device could certainly contribute to the biological control to reduce the aphid infestations. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Augmentative biocontrol is one of the possible biological control strategies that is focused on enhancing the number and/or activity of natural enemies in agroecosystems. This strategy involves mass multiplication and periodic release or introduction of natural enemies in fields (Koul and Dhaliwal, 2003). Indeed, the equilibrium population size and dynamic behavior of many phytophagous insects are largely determined by their natural enemies (Waller, 1987). In this sense, many authors have demonstrated the importance of natural enemies in the regulation of pest populations (Price, 1987; Van Driesche and Bellows, 1996). Augmentation of natural enemies provides a biological solution to pests’ problems
* Corresponding author. Fax: +32 81 62 23 12. E-mail addresses: [email protected], [email protected] (P.D. Leroy). 1049-9644/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2010.05.006
in crops where naturally occurring beneficial organisms fail to respond quickly enough to control populations of pests (King et al., 1993). In the context of integrated pest management, an ideal natural enemy is one that consumes sufficient preys at the right time to maintain a pest population below the economic injury threshold for the crop considered (Michaud and Belliure, 2000). Several predators have been studied as efficient beneficial insects to reduce the aphid damages. The importance of generalist predators in reducing the population density of aphids is widely recognized (Hughes, 1989; Dixon, 1998; Jervis and Kidd, 1996). Among them, Coccinellidae and Chrysopidae are certainly the most documented predators since numerous studies have described predator–prey interactions involving these predators (Powell and Pell, 2007; Volkl et al., 2007; Latham and Mills, 2009). Furthermore, predation by Coccinellidae and Chrysopidaes contributes to the suppression of aphids in several agricultural systems (e.g., potatoes, sugar beets, alfalfa, cotton and wheat) (Coderre, 1999; Barbosa et al., 2008).
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In this study, the hoverfly Episyrphus balteatus (De Geer) (Diptera: Syrphidae) was chosen because this syrphid is considered as the most abundant in agroecosystems and natural habitats in central Europe (Tenhumberg and Poehling, 1995; Colignon et al., 2001), as the most efficient aphid predators (Tenhumberg and Poehling, 1991) but also because this hoverfly is associated with different aphid–plant complexes (Bargen et al., 1998). Data from Rojo et al. (2003) indicate that E. balteatus larvae feed on a large variety of aphid species (234 taxa) with strong evidence for best adaptation to aphids on Gramineae. Also, E. balteatus is the most abundant aphidophagous predator in vegetable crops such as broad beans and carrots (respectively, 70% and 80% of the aphidophagous species) (Colignon et al., 2001, 2002). Episyrphus balteatus larvae are particularly voracious feeders, often eating hundreds aphids during their development. Adults have strong abilities to forage for aphid colonies using vision and semiochemicals released from their preys or prey host–plants (Verheggen et al., 2008, 2009b; Almohamad et al., 2007, 2008, 2009). Furthermore, even first instar larvae can move to new aphid colonies: they are capable of covering about 1 m what allows them to move between plants (Banks, 1968) and even if ladybirds appear to be more active among aphid colonies (Brodsky and Barlow, 1986), it has been shown that syrphid larvae, acting in more restricted area without excessively disturbing aphids, significantly reduce the dispersal of aphids (Niku, 1976) and so the spread of viruses in fields. The aim of this work was to obtain a non-expensive biological control device containing eggs after having artificially induced the syrphid oviposition without using any aphids or host–plants parts. So obtained eggs could be introduced in fields to reduce and control the aphid populations. Because natural insect predators are difficult to maintain in situ and because it is not easy to maintain the natural enemy populations at a sufficiently high number in any given area, the introduction of eggs is advantageous since the emerging larvae directly act in situ. 2. Material and methods 2.1. Rearing plants and insects In a climate-controlled room (16 h light photoperiod; 60 ± 5% RH; 20 ± 2 °C), the host–plants – Vicia faba L. – were grown in 9 8 cm plastic pots containing a mixture of vermiculite and perlite (1/1) and were infested with the aphid Acyrthosiphon pisum Harris, Aphis fabae Scopoli, Megoura viciae Buckton or Myzus persicae Sulzer. In the same climatic conditions, but in a different room, E. balteatus larvae were obtained from a mass-production: the hoverflies were reared with sugar, pollen and water and the oviposition was induced by the introduction of infested host–plants in the rearing-cage (75 60 90 cm) during 3 h. The complete life cycle took place on the host–plants daily re-infested with aphids. Syrphid pupae were provided by Katz Biotech AG (Baruth, Germany). 2.2. Oviposition induction with semiochemicals Several experiments were conducted to obtain syrphid eggs without using host–plants and aphids. First, the following semiochemicals were tested to induce the oviposition: E-(b)-farnesene [the major component of many aphids alarm pheromones, (Francis et al., 2005)], R-(+)-limonene [a common plant monoterpene (Paré and Tumlinson, 1999)] and (Z)-3-hexenol [a green leaf alcohol released by plants in response to mechanical damages or infestations (Paré and Tumlinson, 1999)]. All chemicals were purchased from Sigma–Aldrich (Steinheim, Germany) and had a chemical purity >97% (GC analyses).
The previously cited semiochemicals were tested individually or mixed with each other as follow: E-(b)-farnesene + R-(+)-limonene (10/90; 50/50; 90/10 v/v); E-(b)-farnesene + (Z)-3-hexenol (10/90; 50/50; 90/10 v/v). As proposed by Verheggen and colleagues (2008), a rubber septum was used as a dispenser to release continuously the volatile chemicals. The dispenser was placed into a plastic container (50 cm3) (VWR International) closed with a piece of net for aeration and filled with a 100-ll paraffin oil solution (400 ng/ll final concentration) of the tested chemical. As a positive control, 50 A. pisum aphids (adults on a piece of plant) were placed into containers 24 h before the experiments. 2.3. Oviposition induction with sugars A second set of experiments consisted in the evaluation of the oviposition activity of the main aphid honeydew sugars. Only sucrose, fructose and glucose were tested in this study because the other typical honeydew sugars such as melezitose, melibiose and trehalose are too expensive in the context of a mass-production of syrphid eggs. To do so, solutions of the main honeydew sugars (sucrose (S), fructose (F) and glucose (G)) at 10 g/100 ml and 30 g/100 ml in distilled water (for each sugar), were prepared. For both concentrations, single solutions were tested (S; G; F). Different sugar combinations were also evaluated where sugars were added in the same proportions: S + F; S + G; F + G; S + G + F. A 50 ll volume of these solutions was sprayed onto small plastic lamellas (1 5 cm) that were placed into a plastic container (50 cm3). 2.4. Oviposition induction with natural honeydews The third set of experiments consisted in the evaluation of the oviposition activity of natural honeydew. Natural honeydew was obtained from four aphid species (A. pisum, M. viciae, M. persicae and A. fabae). It was collected on plastic lamellas (1 5 cm) placed under infested V. faba during 24 h. Lamellas covered with 10 mg (mass difference between tarred and honeydew covered lamellas) of honeydew were used to study the syrphid oviposition into plastic containers (50 cm3). In all sets of experiments, one gravid E. balteatus female was introduced into a plastic container and was allowed to lay eggs during 3 h. Gravid females were separated from no-gravid ones when they contained mature eggs easily seen through transparent abdominal pleurites (Sadeghi and Gilbert, 2000). Twenty replications, for each experiment, were performed. These tests were conducted in a climate-controlled room at 22 ± 2 °C and 60 ± 5% RH. The gravid E. balteatus females were 15–20 days old and were deprived of aphids for 24 h before the experiments. 2.5. Biological control device: laboratory and field experiments Syrphid eggs were artificially obtained using semiochemicals, natural honeydew or honeydew sugar solutions as described above. During the laboratory experiments, the biological control device consisted in a plastic lamella (1 5 cm) covered with 5, 10 or 15 eggs. At the beginning of the assay (Day 0), V. faba plants (15–20 cm; 2 leaves) were separately potted and then infested with exactly 50 aphids A. pisum (adults). One biological control device covered with syrphid eggs (lamella) was suspended on the plant. The control consisted in an infested V. faba plant without the biological control device. To evaluate its efficiency, the number of hoverfly larvae and the number of surviving aphids found on the plant were counted after 2, 5 and 7 days. Fifteen replications were performed for each density of eggs. These observations were conducted in a climate-controlled room at 22 ± 2 °C and 60 ± 5% RH.
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The field experiments were conducted from June to August 2008 in a field situated in the Gembloux University farm. The mean daily temperature was 16.7 °C (Min. = 7.0 °C and Max. = 31.1 °C) and the mean daily relative humidity was 78.7% (Min. = 32.2% and Max. = 99.0%). Five parcels (1 m2) were delimited and five V. faba plants (20–30 cm; 4–6 leaves) were planted per parcel. All plants were manually infested with 500 A. pisum (June 2008) or A. fabae (July–August 2008). In four of the five parcels, a lamella comporting 15 syrphid eggs was suspended on each plant on Day 0. The fifth parcel was used as a control. To evaluate the biological control device efficiency, the number of larvae and the number of surviving aphids found on plants were daily counted.
2.6. Statistical analyses One-way analysis of variance (ANOVA) followed by Dunnett’s test (comparison with a control) was used to compare the results with the control. Dunnett’s tests (ANOVA, General Linear Model, Comparison with a control) provide a t-value (tobs) and a P-value. All statistical tests were conducted by using Minitab v.15 for WindowsÒ.
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3.2. Oviposition induction with honeydew sugars Figs. 2 and 3 illustrate the oviposition results obtained with ‘‘artificial honeydews” consisting in solutions of the main honeydew sugars (glucose, fructose and sucrose). Eggs were deposited at random on the plastic lamella and on the walls of the container. Although no significant oviposition was induced with solutions containing 10% of sugar, strong induction is observed with solution containing 30% of either fructose or glucose (tobs = 2.67 and P = 0.049; tobs = 3.06 and P = 0.047, respectively, for fructose and glucose). Sucrose did not induce egg laying behavior at either tested concentrations. When proportionally tested in blends (50– 50 v/v) at the concentration of 30%, all the associations did not allowed to obtain a higher number of eggs than the one noted with the control. 3.3. Oviposition induction with natural honeydews
3. Results
Natural honeydews from four aphid species strongly induced oviposition: in all cases, the number of eggs was higher from the control but only the M. viciae honeydew significantly induced the egg laying (tobs = 3.76 and P = 0.002) (Fig. 4). Once again, eggs were deposited at random on the plastic lamella and on the walls of the container.
3.1. Oviposition induction with semiochemicals
3.4. Biological control device: laboratory experiments
Mean numbers of eggs laid after 3 h in presence of semiochemicals that were used to induce the oviposition are presented in Fig. 1. In all cases, when semiochemicals were tested alone or mixed with each other, eggs were obtained and were randomly deposited in the whole container (not only near the volatile source). The number of eggs significantly differs from those observed with the paraffin oil control (negative control) when the aphid alarm pheromone [E-(b)-farnesene] was applied into rubber septum at a dose of 40 lg (tobs = 9.33 and P = 0.007). Furthermore, a higher number of eggs was laid when the E-(b)-farnesene proportion increased in the blends even if no statistical difference could be noted between the three different proportions. Neither limonene nor (Z)-3-hexenol induced significant oviposition of the hoverfly females. It could also be noted that blends did not significantly increase the ovipositional rate.
Vicia faba plants initially infested with 50 aphids (Day 0) were cleaned from aphid infestations after 1 week when in presence of the biological control device (lamella) comporting 10 or 15 E. balteatus eggs (Fig. 5). The treatment was not efficient with only five syrphid eggs located on the lamella. The mean numbers of surviving aphids significantly differed for the three egg density after the fifth day following the biological control device placement on the plant (tobs = 3.53 and P = 0.001; tobs = 7.09 and P < 0.001, respectively, for the Days 5 and 7 and for the 5 eggs density/tobs = 5.89 and P = 0.001; tobs = 7.09 and P < 0.001, respectively, for the Days 5 and 7 and for the 10 eggs density/tobs = 6.32 and P < 0.001; tobs = 7.09 and P < 0.001, respectively, for the Days 5 and 7 and for the 15 egg density). The number of larvae found on the plants was not equal to the number of eggs introduced with the lamellas (Table 1). Indeed, after five days, the emerging rates were
Fig. 1. Oviposition of E. balteatus females placed into a plastic container during 3 h in presence of semiochemicals (EBF, R-(+)-limonene and/or (Z)-3-hexenol) (Mean number of eggs + SE). ‘‘*” and ‘‘***”, respectively indicate significant difference with the control at P < 0.05 and P < 0.001.
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Fig. 2. Oviposition of E. balteatus females placed into a plastic container during 3 h in presence of sugar solutions (10% or 30% sucrose, fructose, glucose in water). (Mean number of eggs + SE). ‘‘*” and ‘‘***”, respectively indicate significant difference with the control at P < 0.05 and P < 0.001.
Fig. 3. Oviposition of E. balteatus females placed into a plastic container during 3 h in presence of blends of sugar solutions (30% of sucrose, fructose, glucose in water). (Mean number of eggs + SE). ‘‘***” indicates significant difference with the control at P < 0.001.
Fig. 4. Oviposition of E. balteatus females placed into a plastic container during 3 h in presence of natural honeydew from the aphids A. fabae, A. pisum, M. persicae or M. viciae. (Mean number of eggs + SE). ‘‘**” indicates significant difference with the control at P < 0.01.
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Fig. 5. Evolution of the A. pisum aphid populations during 1 week under laboratory conditions in presence of a lamella comporting 5, 10 or 15 eggs of the syrphid E. balteatus and suspended on a V. faba plant (Mean number of aphids + SE). ‘‘***” indicates significant differences with the control at P < 0.001.
Table 1 Cumulative number of E. balteatus larvae counted on V. faba plants and cumulative emergence rate over time (2, 5 and 7 days) (Mean number of larvae ± SE). Number of eggs on the lamella
Day 2 Cumulative number of larvae
Cumulative emergence rate (%)
Cumulative number of larvae
Day 5 Cumulative emergence rate (%)
Cumulative number of larvae
Cumulative emergence rate (%)
5 10 15
1.40 ± 0.19 2.66 ± 0.30 3.80 ± 0.50
28.00 ± 3.44 26.67 ± 4.67 25.33 ± 2.17
2.13 ± 0.30 3.33 ± 0.40 4.53 ± 0.40
42.67 ± 5.86 33.33 ± 2.57 30.22 ± 4.23
2.30 ± 0.45 3.40 ± 0.31 4.50 ± 0.40
46.00 ± 3.56 34.00 ± 6.54 30.00 ± 4.83
42.67 ± 5.86%, 33.33 ± 2.57% and 30.22 ± 4.23%, respectively, for the egg density 5, 10 and 15. 3.5. Field experiments A positive effect of the biological control device was clearly observed on aphid populations of A. fabae and A. pisum (Fig. 6 A, B.) Suspended on a V. faba plant, one lamella comporting 15 syrphid eggs allowed the total aphid elimination after 10 days. The initial population of 500 aphids (Day 0) A. pisum (Fig. 6)(B) or A. fabae (Fig.6)(A) was reduced from the third day (Day 3) but a significant reduction of aphids was observed from Day 8 when compared to the control (V. faba plant without lamella) (tobs = 11.87 and P < 0.001; tobs = 15.25 and P < 0.001, tobs = 17.71 and P < 0.001, respectively, for the Days 8, 9 and 10 and for the aphids A. pisum/tobs = 13.45 and P < 0.001; tobs = 17.56 and P < 0.001, tobs = 19.78 and P < 0.001, respectively, for the Days 8, 9 and 10 and for the aphids A. fabae). Like under laboratory conditions, a low emergence rate was observed outdoor: a maximum of 2.8 ± 1.2 and 3.7 ± 1.4 larvae were present on the plant on Day 10, respectively, for the experiments using A. fabae and A. pisum. 4. Discussion In natural environments, predators and parasitoids are known to oviposit in response to several semiochemical stimuli including (1) host–plants secondary metabolites (VOCs, green leaf volatiles, terpenoids,. . .), (2) prey aggregation, sex and alarm pheromones and (3) prey excretory products (honeydew,. . .) (Flint et al., 1979; Turlings et al., 1990; Turlings and Tumlinson, 1992; Steidle and van Loon, 2003; Verheggen et al., 2008). In this study, using semiochemicals from the host plant [(Z)-3hexenol and R-(+)-limonene] and from the aphids [E-(b)-farne-
Day 7
sene], the hoverfly oviposition was artificially induced inside small containers and on inert surfaces. The major component of the aphids alarm pheromone, E-(b)-farnesene, was the most efficient semiochemical to stimulate the syrphid egg laying. The same observation was realized when a rubber septum containing E(b)-farnesene at 400 ng/lL in paraffin oil was placed on a V. faba plant (Verheggen et al., 2008). Here, eggs were also obtained in response to solutions of the main honeydew sugars, specifically with fructose and glucose at a concentration of 30 g/L. Sucrose, fructose and glucose are the main honeydew sugars (Wäckers, 2000; Yao and Akimoto, 2001) what can explain the higher oviposition rates observed with these sugars even if sucrose did not enhance the number of eggs artificially obtained. It has been suggested that, before the egg laying, syrphids detect sugars using the labellum, tarsal receptors or sensilla on the ovipositor (Hood Henderson, 1982; Hood Henderson and Wellington, 1982). Thought, natural honeydew was the most efficient ovipositional stimulant, confirming that the aphid excretory product is the main factor inducing the syrphid oviposition (Budenberg et al., 1992; Bargen et al., 1998; Sutherland et al., 2001). In this sense, Budenberg and Powell (1992) and Scholz and Poehling (2000) have demonstrated that, on the plant, honeydew induced particular behaviors like searching, localization and oviposition for the syrphids E. balteatus and M. corollae. These results led to the conception of a biological control device consisting in an inert surface (plastic lamella/container) comporting E. balteatus eggs. Using semiochemicals, sugar solutions or natural honeydew, eggs were artificially obtained and then introduced in aphid infested fields by the way of lamellas covered with syrphid eggs. Results obtained were promising since significant aphid populations (500 individuals) were totally eliminated in 10 days with only 15 eggs introduced per plant and even if the emerging rate was very low (<50%). Under laboratory conditions, the number of larvae found on the plant was not equal to the number of eggs
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Fig. 6. Outdoor evolution of the aphid populations (A. fabae (A) or A. pisum (B)) and evolution of the number of the E. balteatus larvae on the plant during 10 days in presence of a lamella comporting 15 eggs of the syrphid E. balteatus and suspended on a V. faba plant (Mean number of aphids + SE). ‘‘***” indicates significant differences with the control at P < 0.001.
introduced with the lamellas. Cannibalism, competition between larvae or eggs consumption by the first emerged larvae (Branquart et al., 1997), could explain the low emerging rates. The same observations were noted outdoor in fields where only few but sufficient larvae reached the plant and were able to eliminate all the aphids. The low recovery rates of larvae on the plants could be explained by the disappearance of some larvae leaving the plants after egg hatch, by cannibalism, by eggs desiccation and/or by starvation. Because female hoverflies sometimes produce sterile eggs, it could certainly be another reason for the low recovery rates of larvae on the plants. In this sense, Chambers (1986) only observed 45% successful hatch for the syrphid Metasyrphus corollae and mainly associated the low viability of eggs to sterility, possibly as a result of insufficient matings. According to this author, desiccation and cannibalism of fertile eggs appeared to be less important. Nevertheless, trials of the present study showed that only few larvae are sufficient to eliminate a significant population of aphids. With this work, we elaborated a biological control device containing or comporting eggs which can easily be introduced in the fields or in greenhouses. According to us, this method constitutes an adequate technique to enhance the hoverfly populations even if the recovery rates of larvae on the plants were low. Indeed, quickly after the emergence, young larvae are able to locate preys
and directly feed on aphids (Gries, 1986; Bargen et al., 1998). Introduction of eggs insures a local treatment since larvae move on short distances and have limited dispersal activity, especially on trichomes-rich plants (Verheggen et al., 2009a). This is in contrast with the adults whose present an high dispersal ability and do not necessarily act on the target area. Furthermore, even if many authors underlined that syrphids lay eggs at the beginning of aphid population build-up (Chambers and Adams, 1986; Tenhumberg and Poehling, 1995) and that hoverfly larvae feed vigorously on aphids, syrphid natural populations are often not sufficient to entirely eliminate aphids’ populations. The introduction of eggs provides a solution to enhance the syrphid populations at the right time, in the right area and in adequate amounts what is not so easy with the techniques currently proposed as the pupae or the adults introduction in the fields. Indeed, pre-reproductive females emerging from pupae are only able to lay fertile eggs after 1 week (Geusen-Pfister, 1987; Sadeghi and Gilbert, 2000), which do not guarantee their presence in the area to protect at the right time. The same problem occurs with gravid females that are inclined to disperse since hoverflies are highly mobile insects (Bondarenko and Asyakin, 1981; Hondelmann et al., 2005). Furthermore, Wyss et al. (1999) observed a high effectiveness of realizing syrphid larvae and eggs in semi-fields experiments and concluded that this
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method appeared to be more suitable than the pupae and adults releases. Further works have to focus on a method that would allow gently application of the eggs in the fields. Like in this work, lamellas covered with eggs can be suspended on plants but a ‘‘pulverization” of eggs obtained on small balls (alginate, chitosan,. . .) could be another approach in order to treat larger surfaces more rapidly and more economically through the use of an air stream treatment as proposed by Shaw and Wallis (2007) to apply a predatory mite in fields. Because we only tested a limited number of treatments on a defined plant and with only one colony size of aphids, same experiments have to be carried out to really assess the effectiveness of this biological control device: introduction of different number of eggs (on the egg carrying plastic lamella), experiments with different plants presenting different structures (trichomes,. . .), experiments considering the distance between the card releasing point and the aphid colonies and experiments with different aphid densities. The use of such a biological control device could certainly enhance the syrphid occurrences in fields and could contribute to the augmentative biocontrol through a natural way of fighting aphids. Firstly, larvae emerging from the introduced eggs will consume the aphids in the targeted area and the resulting adults will search other areas infested with aphids to lay eggs in their turn. Acknowledgments We thank the two anonymous reviewers for their thoughtful comments on an earlier draft of the manuscript. This research was funded by the Walloon Region Ministry Grant (WALEO2: SOLAPHIDRW/FUSAGX 061/6287). References Almohamad, R., Verheggen, F., Francis, F., Haubruge, E., 2007. Predatory hoverflies select their oviposition site according to aphid host plant and aphid species. Entomologia Experimentalis et Applicata 125 (1), 13–21. Almohamad, R., Verheggen, F., Francis, F., Hance, T., Haubruge, E., 2008. Discrimination of parasitized aphids by a hoverfly predator: effects on larval performance, foraging, and oviposition behavior. Entomologia Experimentalis et Applicata 128 (1), 73–80. Almohamad, R., Verheggen, F.J., Haubruge, E., 2009. Searching and oviposition behavior of female aphidophagous hoverflies (Diptera: Syrphidae): a review. Biotechnologie, Agronomie. Société et Environnement 13 (3), 467–481. Banks, C.J., 1968. Effects of insect predators on small populations of Aphis fabae in the field. Entomologia Experimentalis et Applicata 11, 169–176. Barbosa, L.R., de Carvalho, C.F., Souza, B., Auad, A.M., 2008. Efficiency of Chrysoperla externa (Hagen, 1861) (Neuroptera: Chrysopidae) in the Myzus persicae (Sulzer, 1776) (Hemiptera: Aphididae) population reduction in sweet pepper (Capsicum annum L.). Ciencia e Agrotecnologia 32 (4), 1113–1119. Bargen, H., Saudhof, K., Poelhing, H.M., 1998. Prey finding by larvae and adult females of Episyrphus balteatus. Entomologia Experimentalis et Applicata 87, 245–254. Bondarenko, N.V., Asyakin, B.P., 1981. Behaviour of the predatory midge [Aphidoletes aphidimyza Rond.] and other aphidivorous insects in relation to population density of the prey. In: Pristavko, V.P. (Ed.), Insect Behaviour as a Basis for Developing Control Measures against Pests of Field Crops and Forests. Oxonian Press, New Delhi, pp. 6–14. Branquart, E., Hemptinne, J.L., Bauffe, C., Benfekih, L., 1997. Cannibalism in Episyrphus balteatus (Dipt.: Syrphidae). Entomophaga 42, 145–152. Brodsky, L.M., Barlow, C.A., 1986. Escape response of the pea aphid, Acyrthosiphon pisum (Harris) (Homoptera: Aphididae): influence of predator type and temperature. Canadian Journal of Zoology 64, 937–939. Budenberg, W.J., Powell, W., 1992. The role of honeydew as an ovipositional stimulant for two species of syrphids. Entomologia Experimentalis et Applicata 64, 57–61. Budenberg, W.J., Powell, W., Clark, S.J., 1992. The influence of aphids and honeydew on the leaving rate of searching aphid parasitoids from wheat plants. Entomologia Experimentalis et Applicata 63, 259–264. Chambers, R.J., 1986. Preliminary experiments on the potential of hoverflies [Dipt.: Syrphidae] for the control of aphids under glass. Entomophaga 31 (2), 197–204. Chambers, R.J., Adams, T.H.L., 1986. Quantification of the impact of hoverflies (Diptera: Syrphidae) on cereal aphids in winter wheat: an analysis of field populations. Journal of Applied Ecology 23, 895–904. Coderre, D., 1999. Use of aphid-eating ladybugs in biological pest control. Annals of the Entomological Society 35, 14–22.
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Biological Control journal homepage: www.elsevier.com/locate/ybcon
An introduction device for the aphidophagous hoverfly Episyrphus balteatus (De Geer) (Diptera: Syrphidae) Pascal D. Leroy *, François J. Verheggen, Quentin Capella, Frédéric Francis, Eric Haubruge Department of Functional and Evolutionary Entomology, University of Liege, Gembloux Agro-Bio Tech, Passage des Déportés 2, B-5030 Gembloux, Belgium
a r t i c l e
i n f o
Article history: Received 16 November 2009 Accepted 10 May 2010 Available online 31 May 2010 Keywords: Biological control Augmentative biocontrol Aphid Acyrthosiphon pisum Aphis fabae Megoura viciae Myzus persicae Aphidophagous Beneficial Natural enemies Predator Syrphid Episyrphus balteatus Oviposition induction Semiochemicals E-(b)-farnesene R-(+)-limonene (Z)-3-hexenol Honeydew Artificial honeydew
a b s t r a c t Augmentative biocontrol constitutes a safe option to reduce pest populations through the enhancement of natural enemies’ activity. In this context, the aphidophagous syrphid Episyrphus baltetaus (De Geer) (Diptera: Syrphidae) is a promising candidate for aphid biological control: larvae of this syrphid attack and consume a wide range of aphid species and are found on many vegetable crops. Because natural populations of beneficial insects are not always sufficient to regulate the pest infestations, this work has focused on the conception of a biological control device containing syrphid eggs which ones can easily be introduced in fields or greenhouses. Using semiochemicals [E-(b)-farnesene, R-(+)-limonene and (Z)-3-hexenol], honeydews and ‘‘artificial honeydews” (10% or 30% aqueous solutions of sucrose, fructose and glucose), the syrphid oviposition was artificially induced on an inert surface. Specifically, E-(b)-farnesene and concentrated mono-sugars (30%) were identified as the most efficient ovipositional stimulants. To test and validate the biological control device described above, laboratory and field experiments were performed: a plastic lamella covered with syrphid eggs was suspended on aphid infested plants in order to measure the efficiency of the device. The results obtained were promising since populations of 500 aphids were eliminated in 10 days when 15 syrphid eggs were introduced. The use of such a biological control device could certainly contribute to the biological control to reduce the aphid infestations. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Augmentative biocontrol is one of the possible biological control strategies that is focused on enhancing the number and/or activity of natural enemies in agroecosystems. This strategy involves mass multiplication and periodic release or introduction of natural enemies in fields (Koul and Dhaliwal, 2003). Indeed, the equilibrium population size and dynamic behavior of many phytophagous insects are largely determined by their natural enemies (Waller, 1987). In this sense, many authors have demonstrated the importance of natural enemies in the regulation of pest populations (Price, 1987; Van Driesche and Bellows, 1996). Augmentation of natural enemies provides a biological solution to pests’ problems
* Corresponding author. Fax: +32 81 62 23 12. E-mail addresses: [email protected], [email protected] (P.D. Leroy). 1049-9644/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2010.05.006
in crops where naturally occurring beneficial organisms fail to respond quickly enough to control populations of pests (King et al., 1993). In the context of integrated pest management, an ideal natural enemy is one that consumes sufficient preys at the right time to maintain a pest population below the economic injury threshold for the crop considered (Michaud and Belliure, 2000). Several predators have been studied as efficient beneficial insects to reduce the aphid damages. The importance of generalist predators in reducing the population density of aphids is widely recognized (Hughes, 1989; Dixon, 1998; Jervis and Kidd, 1996). Among them, Coccinellidae and Chrysopidae are certainly the most documented predators since numerous studies have described predator–prey interactions involving these predators (Powell and Pell, 2007; Volkl et al., 2007; Latham and Mills, 2009). Furthermore, predation by Coccinellidae and Chrysopidaes contributes to the suppression of aphids in several agricultural systems (e.g., potatoes, sugar beets, alfalfa, cotton and wheat) (Coderre, 1999; Barbosa et al., 2008).
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In this study, the hoverfly Episyrphus balteatus (De Geer) (Diptera: Syrphidae) was chosen because this syrphid is considered as the most abundant in agroecosystems and natural habitats in central Europe (Tenhumberg and Poehling, 1995; Colignon et al., 2001), as the most efficient aphid predators (Tenhumberg and Poehling, 1991) but also because this hoverfly is associated with different aphid–plant complexes (Bargen et al., 1998). Data from Rojo et al. (2003) indicate that E. balteatus larvae feed on a large variety of aphid species (234 taxa) with strong evidence for best adaptation to aphids on Gramineae. Also, E. balteatus is the most abundant aphidophagous predator in vegetable crops such as broad beans and carrots (respectively, 70% and 80% of the aphidophagous species) (Colignon et al., 2001, 2002). Episyrphus balteatus larvae are particularly voracious feeders, often eating hundreds aphids during their development. Adults have strong abilities to forage for aphid colonies using vision and semiochemicals released from their preys or prey host–plants (Verheggen et al., 2008, 2009b; Almohamad et al., 2007, 2008, 2009). Furthermore, even first instar larvae can move to new aphid colonies: they are capable of covering about 1 m what allows them to move between plants (Banks, 1968) and even if ladybirds appear to be more active among aphid colonies (Brodsky and Barlow, 1986), it has been shown that syrphid larvae, acting in more restricted area without excessively disturbing aphids, significantly reduce the dispersal of aphids (Niku, 1976) and so the spread of viruses in fields. The aim of this work was to obtain a non-expensive biological control device containing eggs after having artificially induced the syrphid oviposition without using any aphids or host–plants parts. So obtained eggs could be introduced in fields to reduce and control the aphid populations. Because natural insect predators are difficult to maintain in situ and because it is not easy to maintain the natural enemy populations at a sufficiently high number in any given area, the introduction of eggs is advantageous since the emerging larvae directly act in situ. 2. Material and methods 2.1. Rearing plants and insects In a climate-controlled room (16 h light photoperiod; 60 ± 5% RH; 20 ± 2 °C), the host–plants – Vicia faba L. – were grown in 9 8 cm plastic pots containing a mixture of vermiculite and perlite (1/1) and were infested with the aphid Acyrthosiphon pisum Harris, Aphis fabae Scopoli, Megoura viciae Buckton or Myzus persicae Sulzer. In the same climatic conditions, but in a different room, E. balteatus larvae were obtained from a mass-production: the hoverflies were reared with sugar, pollen and water and the oviposition was induced by the introduction of infested host–plants in the rearing-cage (75 60 90 cm) during 3 h. The complete life cycle took place on the host–plants daily re-infested with aphids. Syrphid pupae were provided by Katz Biotech AG (Baruth, Germany). 2.2. Oviposition induction with semiochemicals Several experiments were conducted to obtain syrphid eggs without using host–plants and aphids. First, the following semiochemicals were tested to induce the oviposition: E-(b)-farnesene [the major component of many aphids alarm pheromones, (Francis et al., 2005)], R-(+)-limonene [a common plant monoterpene (Paré and Tumlinson, 1999)] and (Z)-3-hexenol [a green leaf alcohol released by plants in response to mechanical damages or infestations (Paré and Tumlinson, 1999)]. All chemicals were purchased from Sigma–Aldrich (Steinheim, Germany) and had a chemical purity >97% (GC analyses).
The previously cited semiochemicals were tested individually or mixed with each other as follow: E-(b)-farnesene + R-(+)-limonene (10/90; 50/50; 90/10 v/v); E-(b)-farnesene + (Z)-3-hexenol (10/90; 50/50; 90/10 v/v). As proposed by Verheggen and colleagues (2008), a rubber septum was used as a dispenser to release continuously the volatile chemicals. The dispenser was placed into a plastic container (50 cm3) (VWR International) closed with a piece of net for aeration and filled with a 100-ll paraffin oil solution (400 ng/ll final concentration) of the tested chemical. As a positive control, 50 A. pisum aphids (adults on a piece of plant) were placed into containers 24 h before the experiments. 2.3. Oviposition induction with sugars A second set of experiments consisted in the evaluation of the oviposition activity of the main aphid honeydew sugars. Only sucrose, fructose and glucose were tested in this study because the other typical honeydew sugars such as melezitose, melibiose and trehalose are too expensive in the context of a mass-production of syrphid eggs. To do so, solutions of the main honeydew sugars (sucrose (S), fructose (F) and glucose (G)) at 10 g/100 ml and 30 g/100 ml in distilled water (for each sugar), were prepared. For both concentrations, single solutions were tested (S; G; F). Different sugar combinations were also evaluated where sugars were added in the same proportions: S + F; S + G; F + G; S + G + F. A 50 ll volume of these solutions was sprayed onto small plastic lamellas (1 5 cm) that were placed into a plastic container (50 cm3). 2.4. Oviposition induction with natural honeydews The third set of experiments consisted in the evaluation of the oviposition activity of natural honeydew. Natural honeydew was obtained from four aphid species (A. pisum, M. viciae, M. persicae and A. fabae). It was collected on plastic lamellas (1 5 cm) placed under infested V. faba during 24 h. Lamellas covered with 10 mg (mass difference between tarred and honeydew covered lamellas) of honeydew were used to study the syrphid oviposition into plastic containers (50 cm3). In all sets of experiments, one gravid E. balteatus female was introduced into a plastic container and was allowed to lay eggs during 3 h. Gravid females were separated from no-gravid ones when they contained mature eggs easily seen through transparent abdominal pleurites (Sadeghi and Gilbert, 2000). Twenty replications, for each experiment, were performed. These tests were conducted in a climate-controlled room at 22 ± 2 °C and 60 ± 5% RH. The gravid E. balteatus females were 15–20 days old and were deprived of aphids for 24 h before the experiments. 2.5. Biological control device: laboratory and field experiments Syrphid eggs were artificially obtained using semiochemicals, natural honeydew or honeydew sugar solutions as described above. During the laboratory experiments, the biological control device consisted in a plastic lamella (1 5 cm) covered with 5, 10 or 15 eggs. At the beginning of the assay (Day 0), V. faba plants (15–20 cm; 2 leaves) were separately potted and then infested with exactly 50 aphids A. pisum (adults). One biological control device covered with syrphid eggs (lamella) was suspended on the plant. The control consisted in an infested V. faba plant without the biological control device. To evaluate its efficiency, the number of hoverfly larvae and the number of surviving aphids found on the plant were counted after 2, 5 and 7 days. Fifteen replications were performed for each density of eggs. These observations were conducted in a climate-controlled room at 22 ± 2 °C and 60 ± 5% RH.
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The field experiments were conducted from June to August 2008 in a field situated in the Gembloux University farm. The mean daily temperature was 16.7 °C (Min. = 7.0 °C and Max. = 31.1 °C) and the mean daily relative humidity was 78.7% (Min. = 32.2% and Max. = 99.0%). Five parcels (1 m2) were delimited and five V. faba plants (20–30 cm; 4–6 leaves) were planted per parcel. All plants were manually infested with 500 A. pisum (June 2008) or A. fabae (July–August 2008). In four of the five parcels, a lamella comporting 15 syrphid eggs was suspended on each plant on Day 0. The fifth parcel was used as a control. To evaluate the biological control device efficiency, the number of larvae and the number of surviving aphids found on plants were daily counted.
2.6. Statistical analyses One-way analysis of variance (ANOVA) followed by Dunnett’s test (comparison with a control) was used to compare the results with the control. Dunnett’s tests (ANOVA, General Linear Model, Comparison with a control) provide a t-value (tobs) and a P-value. All statistical tests were conducted by using Minitab v.15 for WindowsÒ.
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3.2. Oviposition induction with honeydew sugars Figs. 2 and 3 illustrate the oviposition results obtained with ‘‘artificial honeydews” consisting in solutions of the main honeydew sugars (glucose, fructose and sucrose). Eggs were deposited at random on the plastic lamella and on the walls of the container. Although no significant oviposition was induced with solutions containing 10% of sugar, strong induction is observed with solution containing 30% of either fructose or glucose (tobs = 2.67 and P = 0.049; tobs = 3.06 and P = 0.047, respectively, for fructose and glucose). Sucrose did not induce egg laying behavior at either tested concentrations. When proportionally tested in blends (50– 50 v/v) at the concentration of 30%, all the associations did not allowed to obtain a higher number of eggs than the one noted with the control. 3.3. Oviposition induction with natural honeydews
3. Results
Natural honeydews from four aphid species strongly induced oviposition: in all cases, the number of eggs was higher from the control but only the M. viciae honeydew significantly induced the egg laying (tobs = 3.76 and P = 0.002) (Fig. 4). Once again, eggs were deposited at random on the plastic lamella and on the walls of the container.
3.1. Oviposition induction with semiochemicals
3.4. Biological control device: laboratory experiments
Mean numbers of eggs laid after 3 h in presence of semiochemicals that were used to induce the oviposition are presented in Fig. 1. In all cases, when semiochemicals were tested alone or mixed with each other, eggs were obtained and were randomly deposited in the whole container (not only near the volatile source). The number of eggs significantly differs from those observed with the paraffin oil control (negative control) when the aphid alarm pheromone [E-(b)-farnesene] was applied into rubber septum at a dose of 40 lg (tobs = 9.33 and P = 0.007). Furthermore, a higher number of eggs was laid when the E-(b)-farnesene proportion increased in the blends even if no statistical difference could be noted between the three different proportions. Neither limonene nor (Z)-3-hexenol induced significant oviposition of the hoverfly females. It could also be noted that blends did not significantly increase the ovipositional rate.
Vicia faba plants initially infested with 50 aphids (Day 0) were cleaned from aphid infestations after 1 week when in presence of the biological control device (lamella) comporting 10 or 15 E. balteatus eggs (Fig. 5). The treatment was not efficient with only five syrphid eggs located on the lamella. The mean numbers of surviving aphids significantly differed for the three egg density after the fifth day following the biological control device placement on the plant (tobs = 3.53 and P = 0.001; tobs = 7.09 and P < 0.001, respectively, for the Days 5 and 7 and for the 5 eggs density/tobs = 5.89 and P = 0.001; tobs = 7.09 and P < 0.001, respectively, for the Days 5 and 7 and for the 10 eggs density/tobs = 6.32 and P < 0.001; tobs = 7.09 and P < 0.001, respectively, for the Days 5 and 7 and for the 15 egg density). The number of larvae found on the plants was not equal to the number of eggs introduced with the lamellas (Table 1). Indeed, after five days, the emerging rates were
Fig. 1. Oviposition of E. balteatus females placed into a plastic container during 3 h in presence of semiochemicals (EBF, R-(+)-limonene and/or (Z)-3-hexenol) (Mean number of eggs + SE). ‘‘*” and ‘‘***”, respectively indicate significant difference with the control at P < 0.05 and P < 0.001.
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Fig. 2. Oviposition of E. balteatus females placed into a plastic container during 3 h in presence of sugar solutions (10% or 30% sucrose, fructose, glucose in water). (Mean number of eggs + SE). ‘‘*” and ‘‘***”, respectively indicate significant difference with the control at P < 0.05 and P < 0.001.
Fig. 3. Oviposition of E. balteatus females placed into a plastic container during 3 h in presence of blends of sugar solutions (30% of sucrose, fructose, glucose in water). (Mean number of eggs + SE). ‘‘***” indicates significant difference with the control at P < 0.001.
Fig. 4. Oviposition of E. balteatus females placed into a plastic container during 3 h in presence of natural honeydew from the aphids A. fabae, A. pisum, M. persicae or M. viciae. (Mean number of eggs + SE). ‘‘**” indicates significant difference with the control at P < 0.01.
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Fig. 5. Evolution of the A. pisum aphid populations during 1 week under laboratory conditions in presence of a lamella comporting 5, 10 or 15 eggs of the syrphid E. balteatus and suspended on a V. faba plant (Mean number of aphids + SE). ‘‘***” indicates significant differences with the control at P < 0.001.
Table 1 Cumulative number of E. balteatus larvae counted on V. faba plants and cumulative emergence rate over time (2, 5 and 7 days) (Mean number of larvae ± SE). Number of eggs on the lamella
Day 2 Cumulative number of larvae
Cumulative emergence rate (%)
Cumulative number of larvae
Day 5 Cumulative emergence rate (%)
Cumulative number of larvae
Cumulative emergence rate (%)
5 10 15
1.40 ± 0.19 2.66 ± 0.30 3.80 ± 0.50
28.00 ± 3.44 26.67 ± 4.67 25.33 ± 2.17
2.13 ± 0.30 3.33 ± 0.40 4.53 ± 0.40
42.67 ± 5.86 33.33 ± 2.57 30.22 ± 4.23
2.30 ± 0.45 3.40 ± 0.31 4.50 ± 0.40
46.00 ± 3.56 34.00 ± 6.54 30.00 ± 4.83
42.67 ± 5.86%, 33.33 ± 2.57% and 30.22 ± 4.23%, respectively, for the egg density 5, 10 and 15. 3.5. Field experiments A positive effect of the biological control device was clearly observed on aphid populations of A. fabae and A. pisum (Fig. 6 A, B.) Suspended on a V. faba plant, one lamella comporting 15 syrphid eggs allowed the total aphid elimination after 10 days. The initial population of 500 aphids (Day 0) A. pisum (Fig. 6)(B) or A. fabae (Fig.6)(A) was reduced from the third day (Day 3) but a significant reduction of aphids was observed from Day 8 when compared to the control (V. faba plant without lamella) (tobs = 11.87 and P < 0.001; tobs = 15.25 and P < 0.001, tobs = 17.71 and P < 0.001, respectively, for the Days 8, 9 and 10 and for the aphids A. pisum/tobs = 13.45 and P < 0.001; tobs = 17.56 and P < 0.001, tobs = 19.78 and P < 0.001, respectively, for the Days 8, 9 and 10 and for the aphids A. fabae). Like under laboratory conditions, a low emergence rate was observed outdoor: a maximum of 2.8 ± 1.2 and 3.7 ± 1.4 larvae were present on the plant on Day 10, respectively, for the experiments using A. fabae and A. pisum. 4. Discussion In natural environments, predators and parasitoids are known to oviposit in response to several semiochemical stimuli including (1) host–plants secondary metabolites (VOCs, green leaf volatiles, terpenoids,. . .), (2) prey aggregation, sex and alarm pheromones and (3) prey excretory products (honeydew,. . .) (Flint et al., 1979; Turlings et al., 1990; Turlings and Tumlinson, 1992; Steidle and van Loon, 2003; Verheggen et al., 2008). In this study, using semiochemicals from the host plant [(Z)-3hexenol and R-(+)-limonene] and from the aphids [E-(b)-farne-
Day 7
sene], the hoverfly oviposition was artificially induced inside small containers and on inert surfaces. The major component of the aphids alarm pheromone, E-(b)-farnesene, was the most efficient semiochemical to stimulate the syrphid egg laying. The same observation was realized when a rubber septum containing E(b)-farnesene at 400 ng/lL in paraffin oil was placed on a V. faba plant (Verheggen et al., 2008). Here, eggs were also obtained in response to solutions of the main honeydew sugars, specifically with fructose and glucose at a concentration of 30 g/L. Sucrose, fructose and glucose are the main honeydew sugars (Wäckers, 2000; Yao and Akimoto, 2001) what can explain the higher oviposition rates observed with these sugars even if sucrose did not enhance the number of eggs artificially obtained. It has been suggested that, before the egg laying, syrphids detect sugars using the labellum, tarsal receptors or sensilla on the ovipositor (Hood Henderson, 1982; Hood Henderson and Wellington, 1982). Thought, natural honeydew was the most efficient ovipositional stimulant, confirming that the aphid excretory product is the main factor inducing the syrphid oviposition (Budenberg et al., 1992; Bargen et al., 1998; Sutherland et al., 2001). In this sense, Budenberg and Powell (1992) and Scholz and Poehling (2000) have demonstrated that, on the plant, honeydew induced particular behaviors like searching, localization and oviposition for the syrphids E. balteatus and M. corollae. These results led to the conception of a biological control device consisting in an inert surface (plastic lamella/container) comporting E. balteatus eggs. Using semiochemicals, sugar solutions or natural honeydew, eggs were artificially obtained and then introduced in aphid infested fields by the way of lamellas covered with syrphid eggs. Results obtained were promising since significant aphid populations (500 individuals) were totally eliminated in 10 days with only 15 eggs introduced per plant and even if the emerging rate was very low (<50%). Under laboratory conditions, the number of larvae found on the plant was not equal to the number of eggs
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Fig. 6. Outdoor evolution of the aphid populations (A. fabae (A) or A. pisum (B)) and evolution of the number of the E. balteatus larvae on the plant during 10 days in presence of a lamella comporting 15 eggs of the syrphid E. balteatus and suspended on a V. faba plant (Mean number of aphids + SE). ‘‘***” indicates significant differences with the control at P < 0.001.
introduced with the lamellas. Cannibalism, competition between larvae or eggs consumption by the first emerged larvae (Branquart et al., 1997), could explain the low emerging rates. The same observations were noted outdoor in fields where only few but sufficient larvae reached the plant and were able to eliminate all the aphids. The low recovery rates of larvae on the plants could be explained by the disappearance of some larvae leaving the plants after egg hatch, by cannibalism, by eggs desiccation and/or by starvation. Because female hoverflies sometimes produce sterile eggs, it could certainly be another reason for the low recovery rates of larvae on the plants. In this sense, Chambers (1986) only observed 45% successful hatch for the syrphid Metasyrphus corollae and mainly associated the low viability of eggs to sterility, possibly as a result of insufficient matings. According to this author, desiccation and cannibalism of fertile eggs appeared to be less important. Nevertheless, trials of the present study showed that only few larvae are sufficient to eliminate a significant population of aphids. With this work, we elaborated a biological control device containing or comporting eggs which can easily be introduced in the fields or in greenhouses. According to us, this method constitutes an adequate technique to enhance the hoverfly populations even if the recovery rates of larvae on the plants were low. Indeed, quickly after the emergence, young larvae are able to locate preys
and directly feed on aphids (Gries, 1986; Bargen et al., 1998). Introduction of eggs insures a local treatment since larvae move on short distances and have limited dispersal activity, especially on trichomes-rich plants (Verheggen et al., 2009a). This is in contrast with the adults whose present an high dispersal ability and do not necessarily act on the target area. Furthermore, even if many authors underlined that syrphids lay eggs at the beginning of aphid population build-up (Chambers and Adams, 1986; Tenhumberg and Poehling, 1995) and that hoverfly larvae feed vigorously on aphids, syrphid natural populations are often not sufficient to entirely eliminate aphids’ populations. The introduction of eggs provides a solution to enhance the syrphid populations at the right time, in the right area and in adequate amounts what is not so easy with the techniques currently proposed as the pupae or the adults introduction in the fields. Indeed, pre-reproductive females emerging from pupae are only able to lay fertile eggs after 1 week (Geusen-Pfister, 1987; Sadeghi and Gilbert, 2000), which do not guarantee their presence in the area to protect at the right time. The same problem occurs with gravid females that are inclined to disperse since hoverflies are highly mobile insects (Bondarenko and Asyakin, 1981; Hondelmann et al., 2005). Furthermore, Wyss et al. (1999) observed a high effectiveness of realizing syrphid larvae and eggs in semi-fields experiments and concluded that this
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method appeared to be more suitable than the pupae and adults releases. Further works have to focus on a method that would allow gently application of the eggs in the fields. Like in this work, lamellas covered with eggs can be suspended on plants but a ‘‘pulverization” of eggs obtained on small balls (alginate, chitosan,. . .) could be another approach in order to treat larger surfaces more rapidly and more economically through the use of an air stream treatment as proposed by Shaw and Wallis (2007) to apply a predatory mite in fields. Because we only tested a limited number of treatments on a defined plant and with only one colony size of aphids, same experiments have to be carried out to really assess the effectiveness of this biological control device: introduction of different number of eggs (on the egg carrying plastic lamella), experiments with different plants presenting different structures (trichomes,. . .), experiments considering the distance between the card releasing point and the aphid colonies and experiments with different aphid densities. The use of such a biological control device could certainly enhance the syrphid occurrences in fields and could contribute to the augmentative biocontrol through a natural way of fighting aphids. Firstly, larvae emerging from the introduced eggs will consume the aphids in the targeted area and the resulting adults will search other areas infested with aphids to lay eggs in their turn. Acknowledgments We thank the two anonymous reviewers for their thoughtful comments on an earlier draft of the manuscript. This research was funded by the Walloon Region Ministry Grant (WALEO2: SOLAPHIDRW/FUSAGX 061/6287). References Almohamad, R., Verheggen, F., Francis, F., Haubruge, E., 2007. Predatory hoverflies select their oviposition site according to aphid host plant and aphid species. Entomologia Experimentalis et Applicata 125 (1), 13–21. Almohamad, R., Verheggen, F., Francis, F., Hance, T., Haubruge, E., 2008. Discrimination of parasitized aphids by a hoverfly predator: effects on larval performance, foraging, and oviposition behavior. Entomologia Experimentalis et Applicata 128 (1), 73–80. Almohamad, R., Verheggen, F.J., Haubruge, E., 2009. Searching and oviposition behavior of female aphidophagous hoverflies (Diptera: Syrphidae): a review. Biotechnologie, Agronomie. Société et Environnement 13 (3), 467–481. Banks, C.J., 1968. Effects of insect predators on small populations of Aphis fabae in the field. Entomologia Experimentalis et Applicata 11, 169–176. Barbosa, L.R., de Carvalho, C.F., Souza, B., Auad, A.M., 2008. Efficiency of Chrysoperla externa (Hagen, 1861) (Neuroptera: Chrysopidae) in the Myzus persicae (Sulzer, 1776) (Hemiptera: Aphididae) population reduction in sweet pepper (Capsicum annum L.). Ciencia e Agrotecnologia 32 (4), 1113–1119. Bargen, H., Saudhof, K., Poelhing, H.M., 1998. Prey finding by larvae and adult females of Episyrphus balteatus. Entomologia Experimentalis et Applicata 87, 245–254. Bondarenko, N.V., Asyakin, B.P., 1981. Behaviour of the predatory midge [Aphidoletes aphidimyza Rond.] and other aphidivorous insects in relation to population density of the prey. In: Pristavko, V.P. (Ed.), Insect Behaviour as a Basis for Developing Control Measures against Pests of Field Crops and Forests. Oxonian Press, New Delhi, pp. 6–14. Branquart, E., Hemptinne, J.L., Bauffe, C., Benfekih, L., 1997. Cannibalism in Episyrphus balteatus (Dipt.: Syrphidae). Entomophaga 42, 145–152. Brodsky, L.M., Barlow, C.A., 1986. Escape response of the pea aphid, Acyrthosiphon pisum (Harris) (Homoptera: Aphididae): influence of predator type and temperature. Canadian Journal of Zoology 64, 937–939. Budenberg, W.J., Powell, W., 1992. The role of honeydew as an ovipositional stimulant for two species of syrphids. Entomologia Experimentalis et Applicata 64, 57–61. Budenberg, W.J., Powell, W., Clark, S.J., 1992. The influence of aphids and honeydew on the leaving rate of searching aphid parasitoids from wheat plants. Entomologia Experimentalis et Applicata 63, 259–264. Chambers, R.J., 1986. Preliminary experiments on the potential of hoverflies [Dipt.: Syrphidae] for the control of aphids under glass. Entomophaga 31 (2), 197–204. Chambers, R.J., Adams, T.H.L., 1986. Quantification of the impact of hoverflies (Diptera: Syrphidae) on cereal aphids in winter wheat: an analysis of field populations. Journal of Applied Ecology 23, 895–904. Coderre, D., 1999. Use of aphid-eating ladybugs in biological pest control. Annals of the Entomological Society 35, 14–22.
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