Introduction

Intraguild predation (IGP) where species that compete for a shared (extraguild) prey also engage in predator–prey interactions (Polis et al. 1989; Holt and Polis 1997) is common in food webs both in natural systems and in managed systems of pests and natural enemies (Polis and Holt 1992; Rosenheim et al. 1995; Janssen et al. 2006). IGP may have a negative effect on the outcome of biological control (e.g. Rees and Onsager 1982; Snyder and Ives 2001; Rosenheim 2005) although some authors have found that the presence of intraguild predators could have a positive effect on pest biocontrol (e.g. Schausberger and Walzer 2001; Snyder et al. 2004; Gardiner and Landis 2007). In biocontrol programmes of greenhouse crops, several species of natural enemies are often used concurrently to combat the pest complex. When polyphagous predators are part of such programmes, the possibility arises that IGP occurs and affects biological control. Optimal combinations of beneficial species for simultaneous use in biocontrol should therefore be based on knowledge on the potential for and intensity of IGP among the various biocontrol agents (Meyling et al. 2004).

Biological control of the sweet potato whitefly, Bemisia tabaci (Gennadius) Biotype B (B. argentifolii Bellows and Perring) (Homoptera: Aleyrodidae) (Oliveira et al. 2001), an economically important pest, is frequently based on the use of aphelinid parasitoids, especially of the genera Encarsia and Eretmocerus (Hymenoptera: Aphelinidae) (Gerling et al. 2001; Naranjo and Ellsworth 2005; Asiimwe et al. 2007), with Encarsia formosa (Gahan) being among the best studied biological control agents of whiteflies. In greenhouse crops, whiteflies often occur alongside thrips, which can be controlled using predatory mites (Riudavets 1995; Zhang 2003) and polyphagous minute pirate bugs of the genus Orius (Heteroptera: Anthocoridae) (e.g. Trottin-Caudal et al. 1991; Tommasini et al. 2004). Among the minute pirate bugs, the species Orius majusculus (Reuter) also has a potential for control of whiteflies (Arno et al. 2008). In spite of the fact that the recently marketed predatory mite Amblyseius swirski (Athais-Henriot) (Acari: Phytoseiidae) has the ability to control both whiteflies and thrips, also in cases of dual infestations (Messelink et al. 2008), other natural enemies of both whiteflies and thrips will likely still be needed for adequate control in some greenhouse crops or under some greenhouse conditions. A combined used of E. formosa and O. majusculus is therefore expected to take place also in the time to come.

Several studies have documented IGP by generalist predators on the specialist parasitoids in the form of parasitised hosts (coincidental IGP, Polis et al. 1989) (Meyling et al. 2004; McGregor and Gillespie 2005; Shiojiri and Takabayashi 2005). The occurrence of IGP by the oligophagous predator Delphastus catalinae (Horn) (Coleoptera: Coccinellidae) on aphelind parasitoids has been documented in several studies (Heinz et al. 1994; Hoelmer et al. 1994; Zang and Liu 2007), with the predator preferring unparasitised whiteflies. Naranjo (2007) similarly reported IGP by three generalist predators, Geocoris punctipes (Say) (Heteroptera: Lygaeidae), Orius insidiosus (Say) (Heteroptera: Anthocoridae), and Hippodamia convergens Guerin-Meneville (Coleoptera: Coccinellidae), on Eretmocerus sp. nr. emiratus, a parasitoid of B. tabaci. However, these predators either preferred parasitised whiteflies or were non-discriminative (Naranjo 2007).

Studies on the predation of O. majusculus on other predators are scarce (Christensen et al. 2002; Jakobsen et al. 2004; Brødsgaard and Enkegaard 2005) and no information regarding IGP by O. majusculus on E. formosa or other parasitoid species has previously been published. With the overall objective to determine whether the combination of these two biological control agents could result in better control of B. tabaci, we tested the discrimination of predators feeding on parasitised and non-parasitised whitefly nymphs and measured predation on various types of prey.

Materials and methods

Plants and insects

Cucumber plants (Cucumis sativus L., cv. Cordoba RZ) were grown in 9 cm plastic pots filled with peat substrate (Mosebrug A/S, Denmark). The pots were placed in insect-proof net-covered cages (68 × 75 × 82 cm) in a climate-controlled greenhouse compartment at 25 °C, 70 % RH, 16:8 L:D. Plants with two fully extended true leaves were used for experiments.

The sweet potato whitefly, originally supplied by Nina Svae Johansen, BioForsk, Norway, was reared on cucumber plants in similar cages and at similar conditions as above. O. majusculus and E. formosa were supplied by EWH BioProduction, Tappernøje, Denmark. All experiments were conducted in climate cabinets at 25 ± 1 °C, 70 ± 5 % RH, 16:8 L:D.

Production of parasitised whiteflies

To obtain parasitised whitefly nymphs, groups of approximately 20–30 adult whiteflies were introduced into clip cages (2 cm diameter) exposed to the lower surface of a cucumber leaf (1–3 clip cages per leaf) on a potted plant. The whiteflies were allowed to oviposit for 24 h and subsequently removed. The plants with whitefly eggs were incubated in a climate-controlled greenhouse compartment at 25 °C, 70 % RH, 16:8 L:D to allow development to the early 4th nymphal stage. Subsequently, groups of 5–6 adult parasitoids were confined in clip cages (4 cm diameter) placed over the leaf areas where the whitefly nymphs were situated. The parasitoids were allowed to parasitise for 24 h. The plants, now harbouring parasitised nymphs, were incubated under similar conditions as above until parasitoid development had progressed to the desired stage (2nd instar, 3rd instar, or pupal parasitoid stage) (Agekyan 1982).

No-choice tests

IGP of O. majusculus on E. formosa was examined separately for 5th instar predator nymphs and adult females. Each predator stage was offered parasitised whitefly nymphs containing parasitoids of either the 2nd, 3rd or pupal stage. Leaves with parasitised whiteflies were taken from the plants described above. For each leaf area with parasitised whiteflies, fifteen whitefly nymphs containing the desired larval or pupal stage of E. formosa were then identified under a binocular stereoscopic microscope and marked by encircling them with a felt pen. Excess whitefly nymphs were removed using an insect pin. Leaf discs harbouring parasitised whitefly nymphs were cut from the leaves and placed in experimental arenas consisting of organdy-vented Petri dishes (5 cm diameter) with a thin layer (2–3 mm) of 1 % agar on the bottom as a substrate to keep the leaf discs fresh. Since O. majusculus could not walk easily on the agar substrate, any agar surface not occupied by leaf discs was covered by pieces of moistened filter paper. Controls consisted of leaf discs with unparasitised 3rd, early 4th and 4th instar whitefly nymphs. These leaf discs were cut from the leaves taken from the whitefly rearing after identifying 15 whitefly nymphs in the desired stage and removing excess nymphs.

One newly moulted 5th instar nymph or one adult female of O. majusculus was individually placed on each leaf disc. The predators were starved for 24 h prior to the experiment by confining them in small boxes (70 cm3) kept in the laboratory (approx. 22 °C). After 24 h, predator nymphs or adults were removed, and numbers of consumed prey were estimated by subtracting the number of undamaged individuals from the total numbers offered. The number of replicates per treatment varied from 15 to 30.

Choice tests

The results from the no-choice tests demonstrated that O. majusculus was able to prey on both parasitised and unparasitised whitefly nymphs in different developmental stages. We were consequently interested in investigating the prey preference of O. majusculus using all possible combinations of the prey items offered in the no-choice experiment. The following prey combinations were used:

  1. (a)

    15 2nd instar parasitoids and 15 unparasitised 3rd instar whiteflies

  2. (b)

    15 3rd instar parasitoids and 15 unparasitised 3rd instar whiteflies

  3. (c)

    15 parasitoid pupae and 15 unparasitised 3rd instar whiteflies

  4. (d)

    15 2nd instar parasitoids and 15 unparasitised early 4th instar whiteflies

  5. (e)

    15 3rd instar parasitoids and 15 unparasitised early 4th instar whiteflies

  6. (f)

    15 parasitoid pupae and 15 unparasitised early 4th instar whiteflies

  7. (g)

    15 2nd instar parasitoids and 15 unparasitised 4th instar whiteflies

  8. (h)

    15 3rd instar parasitoids and 15 unparasitised 4th instar whiteflies

  9. (i)

    15 parasitoid pupae and 15 unparasitised 4th instar whiteflies

The experimental procedures, experimental conditions, and experimental arenas were as described above except that two different kinds of leaf discs were added to each Petri dish—one harbouring parasitised nymphs and one harbouring unparasitised whitefly nymphs in the desired stage. Prey preference was examined separately for 5th instar predator nymphs and adult females. The predators were individually introduced to each Petri dish and the number of prey consumed was recorded as described above. Each combination was replicated 15–17 times.

Data analysis

The data were analysed with generalised linear models. For the no-choice experiments the effect of prey and predator stage together with their interaction was included as fixed effects. It was assumed that the number of individuals were Poisson distributed with an unknown overdispersion, which had to be estimated. The natural logarithm was used as link function. For the choice experiments the effect of prey combination and predator stage together with their interaction was included as fixed effects. It was assumed that the number of individuals chosen was binomially distributed with an unknown overdispersion, which had to be estimated. The logit function was used as link. All analyses were performed using the procedure Glimmix of SAS (SAS Institute Inc 2010).

For analysis of prey preference, Manly’s preference index (Manly 1974) was calculated for each predator stage and each prey combination:

$$ \beta_ {1} = \frac{{\log \left( {e_{1} /A_{1} } \right)}}{{\log \left( {e_{1} /A_{1} } \right) + \log \left( {e_{2} /A_{2} } \right)}} $$

where β1 is the preference for prey type 1, A 1 and A 2 are the number of prey types 1 and 2 offered, and e 1 and e 2 are the numbers of prey type 1 and 2 remaining after the experiment, respectively. The preference index (β) can attain values between 0 and 1, with a β-value higher than 0.5 indicating a preference for prey type 1. In the present study, parasitised B. tabaci was chosen as prey type 1. Manly’s index applies to data originating from experiments where killed prey items are not replaced (Cock 1978; Sherratt and Harvey 1993). Five replicates (out of a total of 277) in which neither of the two prey types offered had been predated were excluded from the analysis of prey preference. Differences between prey preference indices were analysed using two-way ANOVA and the means were separated using Duncan’s multiple range test at P = 0.05 where applicable (SAS Institute Inc 2010). Each preference index was tested for significance (i.e. difference from a value of 0.5) with a one-sample t test with SAS 9.0 (SAS Institute Inc 2010).

Results

No-choice tests

Both adults and 5th instar nymphs of O. majusculus preyed on different unparasitised and parasitised B. tabaci nymphal stages (Table 1). The predation rate was significantly affected by prey stage (F = 16.32, df = 5, 248, P < 0.0001), predator stage (F = 17.57, df = 1, 248, P < 0.0001) and the interaction between prey and predator stage (F = 4.44, df = 5, 248, P = 0.007). Predation of unparasitised as well as parasitised 4th instar B. tabaci nymphs containing 3rd larval instars and pupae of E. formosa by adult predators was significantly higher than predation by 5th instar predator nymphs (P = 0.0023, P < 0.0001 and P = 0.0040, respectively). Adult O. majusculus showed the highest predation on parasitised whitefly nymphs containing 3rd larval instars of E. formosa, while the 5th instars had the highest predation on unparasitised 3rd instar and parasitised B. tabaci nymphs containing 2nd instar parasitoids. Both adults and 5th instar O. majusculus consumed significantly fewer unparasitised 4th instar B. tabaci nymphs than other prey types (P = 0.0003 and P = 0.0205 for adults and 5th instar predators, respectively).

Table 1 Mean number ± SE of different types of prey consumed by adults and 5th instars O. majusculus
Full size table

Choice tests

In the choice tests, predation rates were significantly affected by the prey combination (F = 2.97, df = 8, 254, P = 0.0034) but not by the predator stage (F = 0.00, df = 1, 254, P = 0.98) or by the interaction between prey combination and predator stage (F = 0.63, df = 8, 254, P = 0.75). Both adults and 5th instars of O. majusculus had a clear preference for parasitised over unparasitised whitefly nymphs (Table 2), with all preference indices being significantly different from 0.5 (P < 0.003 and P < 0.0001 for adults and 5th instar predators, respectively), except for the case of 5th instar predators choosing between unparasitised early 4th instar whiteflies and whiteflies containing E. formosa pupae (P = 0.0725). The mean preference indices of adult O. majusculus did not differ significantly among prey combinations, whereas significant differences were apparent for 5th instar predators, with the lowest and highest preference index being observed in the combination of unparasitised early 4th instar B. tabaci and E. formosa pupae and unparasitised 4th instar B. tabaci and 3rd instar E. formosa, respectively. The preferences exhibited by the two stages of the predator in the different prey combinations were not significantly different except in the prey combination with unparasitised 4th instar whiteflies and 2nd instar parasitoids where 5th instar O. majusculus had a slightly higher preference index for E. formosa (P = 0.0294).

Table 2 Mean ± SE number of prey eaten as well as mean preference indices ± SE of O. majusculus for E. formosa (β1) when offered various prey combinations
Full size table

Discussion

The present results confirm that both adults and large nymphs of O. majusculus are able to prey upon unparasitised B. tabaci with consumption rates comparable to those reported by Arno et al. (2008). In the no-choice tests with unparasitised whiteflies both stages of O. majusculus showed very low predation on 4th instar B. tabaci in comparison to predation on 3rd and early 4th instars. This difference might be explained by differences in cuticle thickness and/or cuticular lipids composition among whitefly stages, which is a known physical defence mechanism against predators in B. tabaci as well as in the greenhouse whitefly Trialeurodes vaporariorum Westwood (Neal et al. 1994), making penetration of the whitefly nymph cuticle increasingly difficult for O. majusculus with increasing host age (Gerling 1990).

Orius majusculus did not only prey upon unparasitised whiteflies but also on B. tabaci nymphs parasitised by E. formosa. The intraguild predator characteristics of O. majusculus towards a range of different beneficials (Christensen et al. 2002; Jakobsen et al. 2004; Brødsgaard and Enkegaard 2005; Sanderson et al. 2005) has herewith been documented to pertain also to parasitoids. IGP on immature stages of parasitoids has been reported for a number of other predators (Hoelmer et al. 1994; Heinz et al. 1994; Heinz and Nelson 1996; Colfer and Rosenheim 2001; Snyder and Ives 2001; Zang and Liu 2007; Naranjo 2007).

In the no-choice tests with parasitised whiteflies both adults and 5th instars of O. majusculus had a lower predation on E. formosa pupae compared with parasitoids in the larval stages. The same pattern has been found for other predators (e.g. Hoelmer et al. 1994; Heinz and Nelson 1996; Al-Zyoud and Sengonca 2004; Fazal and Xiang 2004; Zang and Liu 2007; Kutuk et al. 2011). This phenomenon could be due to changes in chemical and physical characteristics during the pupal phase of parasitoid development (Hoelmer et al. 1994). In addition, parasitism-induced hardening of the whitefly cuticle and formation of air spaces around the developing parasitoid hinders the predator in attacking the parasitoid (Hoelmer et al. 1994; Kutuk et al. 2011). In other parasitoid-predator systems, however, the predation rate is independent of the parasitoid development, or may be highest on parasitoid pupal stages (Naranjo 2007).

In the present study, O. majusculus exhibited a clear preference for parasitised over unparasitised prey, except when 5th instar predators were offered a choice between early 4th instar whiteflies and E. formosa pupae. Similar preferences have been reported for several other predator species (Naranjo 2007). The same author speculated that this preferential predation is related to prey appearance, differential distribution of parasitised and non-parasitised prey or nutritional aspects (Naranjo 2007). Based on our observations, parasitoid development causes the otherwise flat and translucent whitefly nymphs to swell and become opaque (Gelman et al. 2002). This may render the parasitised hosts more apparent to searching O. majusculus.

The ability of O. majusculus to prey upon parasitised B. tabaci and its preference for parasitised hosts may have detrimental effects on E. formosa-based biological control of B. tabaci and, most likely, other whiteflies. Disruption of parasitoid-based biological control by inclusion of an intraguild predator has been documented by several authors (e.g. Rees and Onsager 1982; Raymond et al. 2000; Snyder and Ives 2001). In contrast, Heinz and Nelson (1996) and Colfer and Rosenheim (2001) found that the level of suppression of pest populations was increased despite the occurrence of IGP.

In view of the possible detrimental effects of O. majuscules on E. formosa, the consequences of using O. majusculus against whiteflies (Arno et al. 2008) concurrently with the use of E. formosa should be evaluated based on studies of biocontrol with combinations of these two species. In situations in which O. majusculus is applied for control of other pest species, typically thrips, predation on whiteflies is likely to occur to some extent (Montserrat et al. 2000). Studies on the prey preference of O. majusculus for its target pest and parasitised and unparasitised whiteflies will be needed, as will larger-scale studies in multi-species biocontrol systems under semi-field or field conditions on the degree of IGP on E. formosa and its effect on biocontrol of whiteflies and other pests.