Introduction

The development of synthetic insecticides in the mid-twentieth century made the controlling of herbivorous pests quite adequate, and these chemicals are still widely used in modern agriculture (Lu et al. 2012; Goulson 2019). However, long-term and over-dosage applications of chemical pesticides lead to food safety concerns, insect pest resistance, and adverse effects on non-target organisms (e.g., see Menail et al. 2020; Palma-Onetto et al. 2021; Passos et al. 2022 and Desneux et al. 2007 for a thorough review). Due to these critical limitations, biological control of arthropod pest populations by natural enemies has become an essential pest management method (Ragsdale et al. 2011; Heimpel and Mills 2017; Michaud 2018; Zang et al. 2021) notably in Integrated Pest Management (IPM) packages (Desneux et al. 2022). Biological control is an environmentally friendly technique of using organisms to reduce pests and their effects on crops (Heimpel and Mills 2017). Augmentative biological control—inundation type—involves the release of mass-reared natural enemies, such as predators, in large numbers to obtain immediate pest control (Collier and Van Steenwyck 2004; Perez-Alvarez et al. 2019; Huang et al. 2020). Although augmentative biological control is effective, natural enemies tend to rapidly disappear from crops once pest prey resources are mostly consumed, requiring frequent and costly reintroductions (Bennison et al. 2011; Messelink et al. 2014; Oveja et al. 2016; Labbé et al. 2018). To avoid these challenges, providing alternative diets such as alternative prey and/or companion plants showed great potential for sustaining biocontrol agents and thus to support the biocontrol services they provide (Lundgren et al. 2009; Messelink et al. 2014; Damien et al. 2020; Li et al. 2021). Indeed, food resources from companion plants (e.g., nectar or pollen) and alternative prey (e.g., Ephestia kuehniella eggs) are used as supplemental food to support beneficial arthropods in crops (Messelink et al. 2014).

Companion plants are secondary plants (considering the crop plant as the primary one in the agroecosystem) purposely established directly next to individual crop plants in order to offer an additional service. For instance, whereas crop plants provide food or fiber provision, companion plants provide pest regulation (Parolin et al. 2012a, b; Parker et al. 2013). Several strategies can provide predatory insects with the resources they need (so-called non-prey food or banker plants): food sources (e.g., nectar or pollen) and/or shelter (refuges, habitats, mating or oviposition sites) (Lundgren et al. 2009; Wäckers and Van Rijn 2012; Gurr et al. 2016; Lu et al. 2014; Ramsden et al. 2015; Chen et al. 2022). Then, companion plants allow natural enemies to survive and reproduce within a cropping system which can increase their abundance and their capacity to regulate pest populations (Begum et al. 2006; Frank 2010; Huang et al. 2011; Parolin et al. 2012a, b; Parker et al. 2013; Han et al. 2022). The flowering plant alyssum Lobularia maritima (Brassicaceae) shows several characteristics that could make it an excellent candidate as companion plant. For instance, this plant has a long flowering period (about 10 months) which means it can provide pollen and floral nectar for extended periods (Picó and Retana 2000). It attracts several natural enemies and enhances their fitness, thus having the potential to be an efficient companion plant in various contexts (e.g., Berndt and Wratten 2005; Hogg et al. 2011; Pumariño and Alomar 2012; Tiwari et al. 2020); this flowering plant may sustain natural enemies even when pest populations are low (Messelink et al. 2014).

Artificial food sources are foods produced from one or more ingredients, that are provided to replace natural food (prey or host), and that that are more convenient from a technical and/or economic perspectives (Pascacio-Villafán et al. 2017). Many artificial food sources, such as alternative prey, have the potential for enhancing establishment of natural enemies (Riddick 2009; Messelink et al. 2014). Due to their high percentage of amino acids and lipids, lepidopteran eggs, such as those of moth E. kuehniella (Lepidoptera: Pyralidae), are commonly used for mass rearing of various natural enemies, including predatory heteropterans (e.g., Orius spp., Macrolophus pygmaeus Wagner), coccinellid beetles (e.g., Harmonia axyridis Pallas, Adalia bipunctata L.), lacewings (Chrysoperla spp.), and egg-parasitoids belonging to the Trichogramma genus (Specty et al. 2003; Bonte and De Clercq 2008, 2011; Riddick 2009; St-Onge et al. 2014; Pehli̇van 2021; Zang et al. 2021; Zhang et al. 2021). Cocuzza et al. (1997) proposed E. kuehniella eggs as the most important food source, which resulted in positive effects for O. laevigatus fitness parameters such as fecundity, oviposition period, and female longevity.

The simultaneous use of various alternative food resources has been proposed to induce synergistic positive effects on predator population growth (Pekas and Wäckers 2017); combination of multiple resources for mutualistic arthropods can be better than the sum of each reward provided individually. Likewise, the combination of the companion plant L. maritima  (alyssum thereafter) together with the alternative prey Ephestia eggs may have an additive, or even so a synergistic, enhancing effect(s) on predator population growth.

In this context we  aimed to characterize the effects of supplemental food resources on establishing O. laevigatus populations in strawberry cropping systems. The study was designed to (i) determine whether a strawberry crop alone can support O. laevigatus population growth, (ii) test if the addition of alyssum as an intercrop to strawberry can enhance O. laevigatus population growth, (iii) determine if adding Ephestia eggs can enhance O. laevigatus population growth, (iv) assess if alyssum combined with Ephestia eggs can support increased O. laevigatus population growth, owing to an additive or synergistic effect, (v) analyze the effects of these different treatments on the regulation of aphid population on strawberry, and (vi) evaluate the potential impact on crop yield.

These objectives were tested on the cultivated strawberry Fragaria x ananassa, an important fruit crop. It has grown in popularity in recent decades, as more emphasis is placed on the health benefits of eating fruits and berries (Aaby and Remberg 2015). Nevertheless, Western Flower Thrips (Frankliniella occidentalis) and aphids (e.g., Macrosiphum euphorbiae) are the two major pests in greenhouse strawberry production systems (Solomon et al. 2001; Hulle et al. 2020). The minute pirate bug O. laevigatus (Fieber) (Hemiptera: Anthocoridae) is the most successful biocontrol species in strawberry cropping systems and it is used in augmentative release programs (Bouagga et al. 2018). Its prey is diverse, with a clear preference for pests, including aphids, mites, and thrips (Venzon et al. 2002).

Material and methods

Biological material

Strawberry plants (three-month old seedlings, < 7 cm height), variety 'Amandine' (Pépinières Martaillac, Saint Marthe, France) were planted in 14-cm-diameter pots with potting mix (Sphagnum peat moss, Softwood bark compost, Brown peat, Horse manure, Sea algae, Earthworm Compost) and pine bark. They were placed in a greenhouse for three weeks and supplied with water three times a week (due to high water retention of the potting mix). In addition, a nutrient solution was supplied through capillary systems before and during the experiment.

Lobularia maritima (L.) Desv. 1815 (alyssum) seeds were planted in 7 × 7 × 6.5 cm pots and placed in a growth chamber (25 ± 1 °C, 65 ± 5% RH, and 14:10 L:D). After 20 days, they were transferred to the greenhouse. They were watered and acclimatized for 7 days before being transplanted to larger plastic pots (diameter 10 cm, height 9 cm). The plants had all reached the flowering stage before their use in the experiment.

Orius laevigatus colony originated from Biobest, France, and was reared in plexiglass cages and fed with E. kuehniella eggs on strawberry and alyssum plants. The rearing cages were kept in growth chambers (25 ± 1 °C, 65 ± 5% RH, and 14:10 L:D). The M. euphorbiae colony was initiated using individuals naturally infecting tomato plants in the INRAE Sophia Antipolis experimental station. The aphid colony was reared on tomato plants in greenhouse cages covered with mesh. E. kuehniella eggs used as alternative prey were refrigerated at 8–10 °C for conservation.

Experimental design

Experiments were conducted in an environment-controlled greenhouse (T=25 °C and RH=60%) at INRAE Sophia Antipolis experimental station in Spring 2021. The experimental design was a randomized block design within four compartments comprising four treatments in each compartment (Fig. 1; Suppl. 1). Each of the four distinct compartments in the greenhouse was 40 m2. Treatments in a given compartment were isolated from one another by fine mesh nylon, which formed tunnels (height: 2 m, width: 1 m, length: 5 m) and prevented insect transfer (Jaworski et al. 2015; Campos et al. 2020). Each tunnel contained one row (4.84 m long and 30 cm wide for one row) on which all the potted plants were placed about 20 cm from the soil (Suppl. 1).

Fig. 1
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Greenhouse experimental setup

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The four treatments (A–control; B–alyssum; C–Ephestia eggs and D–alyssum + Ephestia eggs) were arranged in a randomized complete block design and repeated once in each compartment (n = 4; Fig. 1). The strawberry plants were selected to obtain the same number of flowers in each treatment. Treatments without alyssum had 16 strawberry plants with 30 cm between plants, while treatments with alyssum had 16 strawberry plants and 8 alyssums, i.e., one alyssum between 2 strawberry plants. The spacing between strawberry and alyssum plants was 15 cm as recommended by Brennan (2016). Ephestia eggs (1 g/plant) were introduced once a week through sprinkling by hand over strawberry leaves on treatments containing Ephestia eggs.

The predator O. laevigatus and the aphid M. euphorbiae were introduced in all treatments. Before inoculation, aphid individuals were collected from the colony on fresh tomato leaves. Then, 5 randomly selected individuals were placed in every second strawberry plant throughout the four treatments in all compartments (Fig. 1). Aphids were released 1 week before predators. Four O. laevigatus adults (1 male for 3 females) were released 3 times (once a week for the first three weeks) in each replicate for all the treatments in the middle of each row to ensure a homogeneous distribution.

Data recording

Population dynamic of both insect species (O. laevigatus and M. euphorbiae) was monitored weekly during 8 weeks following the first release of predators. The number of individuals was counted in the morning (7:00 to 10:00 a.m.) on each replicate. For each counting, numbers of O. laevigatus (adults and nymphs), and M. euphorbiae (adults and nymphs) per plant (both strawberry and alyssum) were assessed in situ by examining the stems, leaves, and flowers of plants. In addition, strawberry flowers were examined in situ for O. laevigatus nymphs, particularly between the receptacle and the calyx. The number of fruits per strawberry plant was also recorded and used as a proxy for strawberry yield at the end of the experiment.

Statistical analysis

In order to compare the effect of our four treatments on the population dynamic of both the predators and pests, we completed a generalized estimating equation (GEE, package “geepack”) and a Poisson distributed response. The GEE was used to account for the effect of time and data interdependence, as well as the option of including the plant’s identity number in the formula to specify the presence of autocorrelation on the data over time. When significant effects were found, the Least Square Means (“lsmeans”) R package was used to perform Tukey’s HSD tests for post hoc pairwise comparisons. This model enabled us to compare the tendency of the complete temporal set of data between treatments.

The effect of treatments on fruit mean number per treatment was evaluated using ANOVA, associated with the Tukey’s HSD tests for post hoc pairwise comparisons.

All statistical analyses were performed using version 3.4.2 of the R software program (R core Team, 2013).

Results

Impact of treatments on predator population dynamics

The average number of O. laevigatus (nymphs and adults) per plant varied over time and according to the treatments applied (Table 1, Fig. 2). The evolution of the number of predators (nymphs and adults) was almost stable until day 28 before increasing exponentially for the treatments in which alyssum and/or E. kuehniella eggs were included except for the control treatment where the population remained very low (less than 0.5 individuals/plant) from the beginning to the end of the experiment. The presence of alyssum alone and its association with the addition of the eggs seemed to influence the fluctuation of the predator population in a similar way. The number of nymphs was higher when eggs were provided as alternative prey on strawberry plants from day 42.

Table 1 Number of Orius laevigatus adults and nymphs, as well as Macrosiphum euphorbiae (adults + nymphs), per plant (means and SE), according to the treatments and the times (days) as well as all the pairwise interaction (a - GEE: generalized estimating equation, b - Tukey’s HSD post hoc test). For a given group (i.e. O. laevigatus nymphs, O. laevigatus adults, and M. euphorbiae) letters indicate significant differences among treatments and among sampling days (at P < 0.05).
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Fig. 2
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Evolution of O. laevigatus nymphs (a) and adults (b) populations (means and SE) according to the treatments and the times (days)

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Impact of treatments on pest population dynamics

Both treatment and time (and their interaction) have effects on the number of M. euphorbiae individual per plant (Table 1, Fig. 3). The presence of companion plant (alyssum) and alternative prey (E. kuehniella eggs) significantly decrease the number of pest individuals per plant. Without alyssum and E. kuehniella eggs (control treatment), this number increased rapidly from less than 1 individual/plant to more than 7 individuals/plant. The impact of E. kuehniella eggs on M. euphorbiae population was observed only from day 21, with a significant and rapid decrease in M. euphorbiae populations until day 56. On the other hand, in the presence of alyssum (with or without eggs), M. euphorbiae numbers remained consistently low (< 2 individuals/plant).

Fig. 3
figure 3

Evolution of M. euphorbiae (adults + nymphs; mean ± SE) population according to the treatments and the time (days)

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Impact of treatments on fruit production

The average number of fruits produced per plant was influenced by the various treatments applied, i.e., the presence or not of alyssum, the presence or not of E. kuehniella eggs, or the simultaneous presence of both (alyssum + E. kuehniella eggs) (Fig. 4). Analysis of variance indicated significant differences between the number of fruits obtained per plant according to treatments (df = 3, F = 150, P < 0.001). The highest number was obtained when alyssum was associated with the alternative prey, followed, respectively, by the treatments with the presence of alyssum and E. kuehniella eggs only, and the lowest number of fruits was obtained in the control treatment.

Fig. 4
figure 4

The average number of fruits per strawberry plant (mean ± SE) according to the treatments. Different letters indicate significant difference at P < 0.05 (ANOVA followed by Tukey’s HSD post hoc test)

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Discussion

Our study evaluated the importance of providing additional and diversified food sources for the establishment of the predator O. laevigatus population using alternative prey and/or companion plants to control population of M. euphorbiae.

O. laevigatus population was not able to establish on strawberry alone (control); such low level observed could be explained by the fact that the presence of prey (aphids) only on strawberry is not qualitatively and/or quantitatively sufficient to allow the predator establishment and growth (Desneux et al. 2019). This could also be due to this species’ difficulty or slowness in establishing and multiplying on strawberry. Indeed, Bennison et al. (2011) asserted that O. laevigatus established slowly on strawberry, especially when flowers are scarce. Regarding M. euphorbiae (aphid) population dynamic in the control treatment, the rapid and continuous increase in aphid number per plant could be related to the low number of predators in this treatment. Indeed, the establishment and very slow growth of predators on strawberry in the absence of alyssum and E. kuehniella eggs could favor the development of aphid populations. Finally, our study's evolution of the number of fruits per plant was inversely related to the number of aphids. When there are more aphids per plant, there are fewer fruits per plant. This result is in line with Paranjpe et al. (2003) where the cultivar with the highest number of fruits showed the lowest number of aphids. Indeed, aphids are known to affect crop yield through the transmission of viruses and/or by sucking out plant sap (Rabasse et al. 2001; Martin and Tzanetakis 2006). However, it should be noted that the conditions for fruit development are not optimal in this experimental design because the strawberry plants were inserted in tunnels closed off by insect-proof nets preventing the monitored insects from getting out but also the external insects, thus the pollinators, from getting in.

The supplementation with Ephestia eggs (Strawberry + Ephestia eggs) increased fecundity and established the highest O. laevigatus population which is in line with a previous laboratory experiment, where we reported an increased number of O. laevigatus nymphs on strawberry supplemented with Ephestia eggs (Suppl. 2). Pumarino and Alomar (2012) also observed a strong positive effect of Ephestia eggs input to another Orius species in laboratory conditions. Providing diets of only plant parts to omnivorous predators is insufficient to maintain a good fitness and the presence of a prey (targeted pest or others) is a necessity for predator fecundity (Vacante et al. 1997; Cocuzza et al. 1997; Pumariño and Alomar 2012; Suppl. 2). Ephestia eggs are also successfully used for mass rearing of O. laevigatus as they have been shown to be nutritionally superior to other natural and artificial foods increasing longevity, especially for the genus Orius (Cocuzza et al. 1997; Pehli̇van 2021; Gallego et al. 2022). Specty et al. (2003) also showed that the predator Harmonia axyridis had a good developmental capacity when fed with E. kuehniella eggs than with aphids as prey but Sylla et al. (2016) showed equivalent effect of both Ephestia and aphid prey on several traits (including fertility) of the mirid predator Macrolophus pygmaeus. On the other hand, the number of aphids in the presence of E. kuehniella eggs dropped later, compared to the control treatment, probably due to the preference of predators to feed first on E. kuehniella eggs which is a better food source (Pehli̇van 2021) before attacking aphids. In a similar experiment on pepper plants, Messelink et al. (2015) also showed that the most efficient control of the aphid population was achieved with Macrolophus pygmaeus as predator in combination with a weekly application of supplemental food including E. kuehniella eggs.

Alyssum could be considered a companion plant of choice in maintaining and growing O. laevigatus populations because it flowers prolifically and continuously provides pollen and nectar for predators, then O. laevigatus could reproduce, even without prey (Bennison et al. 2011; Ribeiro and Gontijo 2017). Alyssum might provide resource subsidies during times of prey scarcity as omnivorous predators may profit from a combination of plant components (particularly pollen; Vacante et al. 1997; Pumariño and Alomar 2012). Furthermore, the reduction of pests over time in the presence of alyssum only or alyssum + E. kuehniella eggs would thus be explained by the negative impact of predator population growth on their growth. In other words, the more O. laevigatus multiplies, the less the number of aphids increases. In effect, once established on alyssum, predators would then disperse on strawberry plants to attack the pests reducing the number of pests, as Bennison et al. (2011) observed in their study on western flower thrips.

A synergistic impact of multiple resources on the population dynamics of predatory mites for better pest control has been demonstrated in citrus (Pekas and Wäckers 2017). In the present study, we expected the treatment [strawberry + alyssum + Ephestia eggs] would provide a more well-balanced and nutrient-rich diet to support Orius populations, as suggested by Vacante et al. (1997) or Pumarino and Alomar (2012) results in laboratory conditions. In addition to benefit from the presence of prey (aphids and/or E. kuehniella eggs), predators also benefit from the presence of pollen from strawberry and alyssum flowers. However the presence of multiple resources was not synergistic even so additive in establishing Orius population when compared to the treatment with alyssum only. Also, as the number of offspring is limited by the initial number of individuals introduced, it is possible that synergistic and/or additional effects of nutrient resources cannot be observed in our conditions. Then to confirm the absence/presence of a synergistic effect, we should redo the experiments by testing higher initial predator density. Interestingly, the highest fruit number was reached in the treatment [strawberry + alyssum + Ephestia eggs] suggesting that pest control could improve strawberry yield, even though this experiment does not demonstrate the effects of aphids on fruit quality (Van Oystaeyen et al. 2022). Indeed, the increase in O. laevigatus populations due to alyssum and E. kuehniella eggs would have reduced aphid damages on strawberry plants by reducing their populations.

The establishment of O. laevigatus populations appears to be a critical factor in their practical use as a biological control agent in strawberry crops. The use of alternative prey and/or companion plant in IPM is being increasingly studied (Letourneau et al. 2011; Biondi et al. 2016; Konan et al. 2021). Moreover, our study provided evidence of the beneficial effects of their use on O. laevigatus and the biological control of M. euphorbiae. Thus, alyssum plants used alone or combined with E. kuehniella eggs-maintained predator populations and reduced M. euphorbiae damage on strawberry plants. These resources, besides providing quality food sources, did not interfere with the control of M. euphorbiae by O. laevigatus and could therefore be considered essential elements in implementing IPM systems for strawberry. Furthermore, the use of alyssum could provide an additional service to the agrosystem, by also attracting pollinators and thus promoting the pollination of strawberry plants (Hodgkiss et al. 2019). With the establishment of the predator population achieved, it is now necessary to question the possibility of maintaining the predator in the system, especially after the collapse of the pest population (Symondson et al. 2002; Messelink et al. 2014). Are these proposed alternative feeds sufficient to maintain the predator population, even in the complete absence of prey?

Author contributions

MZ, CN, AVL, and ND conceived and designed research. MZ, CN, KAJKL, EAD, and PB carried out the experiments. MZ and AVL analyzed data. MZ, CN and AVL wrote the manuscript. All authors reviewed the manuscript.