Effects of far-red light on the behaviour and reproduction of the zoophytophagous predator Macrolophus pygmaeus and its interaction with a whitefly herbivore

Authors

Davy Meijer¹, Syb Hopkoper¹, Berhane Tekle Weldegergis¹, Wendy van ’t Westende², Joop Johannes Antonius van Loon¹, Marcel Dicke¹

¹Laboratory of Entomology, Wageningen University, PO Box 16, NL-6700, AA Wageningen, the Netherlands;²Laboratory of Plant Breeding, Wageningen University, PO Box 16, NL-6700, AA Wageningen, the Netherlands

Abstract

Plants can detect neighbouring plants through a reduction in the ratio between red and far-red light (R:FR). This provides a signal of plant-plant competition and induces rapid plant growth while inhibiting defence against biotic stress, two interlinked responses designated as the shade avoidance syndrome (SAS). Consequently, the SAS can influence plant-herbivore interactions that could cascade to higher trophic levels. However, little is known on how the expression of the SAS can influence tritrophic interactions. We investigated whether changes in R:FR affect the emission of herbivore-induced plant volatiles (HIPVs), and whether these changes influence the attraction of the zoophytophagous predator Macrolophus pygmaeus . We also studied how the expression of the SAS and subsequent inhibition of plant defences affects the reproduction of M. pygmaeus in both the presence and absence of the greenhouse whitefly (Trialeurodes vaporariorum ) as arthropod prey. The results show that changes in R:FR have little effect on HIPV emissions and predator attraction. However, a reduction in R:FR leads to increased reproduction of both the predator and the whiteflies. We conclude that shade avoidance responses can increase the population development of M. pygmaeus directly by reducing plant defences, and indirectly by supporting higher herbivore densities.

Keywords: shade avoidance, volatiles, tritrophic interactions, predator-prey interactions, arthropod performance, Trialeurodes vaporariorum

Introduction

Plants possess elaborate mechanisms to sense and respond to changes in their dynamic environment. These mechanisms help plants to differentially allocate their resources to many physiological and developmental processes in order to adequately respond to the risks and challenges posed by their environment. These allocation patterns often come down to trade-offs between growth and defence (Zust and Agrawal, 2017). An example of such a trade-off is the shade avoidance syndrome (SAS). The SAS is aimed at avoiding shade and light competition from neighbouring plants and consists of morphological and physiological adaptations that stimulate growth and reproduction (Casal, 2012), but it is also associated with reduced defensive responses towards biotic stress (Ballaré, 2014). Over the past decades, there has been increasing interest in understanding how plants perceive and respond to shading by neighbouring plants (Ballaré & Pierik, 2017; Fernández-Milmanda & Ballaré, 2021), and how these responses interact with responses to other biotic and abiotic stresses (Courbier & Pierik, 2019; Lazzarin et al. , 2021). These studies have resulted in valuable insights into how plants balance responses between different stressors, and thereby provide opportunities to improve plant resilience in agricultural settings.

Shade avoidance depends on the detection of far-red light, which is reflected from vegetative plant tissue and therefore provides a signal of plant density and the degree of competition for light (Ballaréet al. , 1990). Reflection of far-red light lowers the ratio between red and far-red light (R:FR), which is detected by the phytochrome B (phyB) photoreceptor. Red light activates phyB, which is then transported to the nucleus and inhibits SAS responses. With increasing far-red light, the R:FR drops and phyB is inactivated, allowing for the expression of SAS (Ballaré and Pierik, 2017). The SAS is characterised by morphological adaptations such as stem elongation, upward leaf movement and reduced branching, which serve to increase light capture in crowded canopies (Casal, 2012). These adaptations are mediated through far-red induced stimulation of the growth-promoting phytohormones auxin and gibberellin (Ballaré and Pierik, 2017). At the same time, inactivation of phyB inhibits the signalling of jasmonic acid (JA) and salicylic acid (SA), the two principal phytohormones involved in defence against biotic stress (Ballaré, 2014; Fernández-Milmanda & Ballaré, 2021). Consequently, plants exposed to far-red light are more susceptible to biotic stress, leading to the increased performance of arthropod herbivores (Izzaguirre et al. , 2006; Moreno et al. , 2009; Cortés et al. , 2016; Meijer et al. , 2022).

Both JA and SA are known to inhibit the signalling of growth-promoting phytohormones (Wasternack and Feussner, 2018; van Butselaar and van den Ackerveken, 2020). Downregulation of defensive signalling is therefore required for the full and rapid expression of SAS, and serves to prioritize SAS responses over defensive responses when experiencing competition for light (Ballaré and Austin, 2019). There are also indications that plants can compensate for the reduced direct defences against arthropod herbivores by stimulating interactions with the natural enemies of the herbivores (Cortés et al. , 2016). Plants that are attacked by herbivores emit herbivore-induced plant volatiles (HIPVs), a blend of volatile organic compounds (VOCs) that attract the natural enemies of the attacking herbivores (Dicke and Baldwin, 2010). These volatile blends are highly specific and can contain information on the herbivore species, density and on the abiotic conditions of the plant (Aartsma et al. , 2017; Cabedo-López et al. , 2019). Exposure to far-red light can influence VOC emissions, with consequences for plant-arthropod interactions (Kegge et al. , 2013; Cortéset al. , 2016). Plants treated with methyl jasmonate (MeJA), a volatile form of the stress hormone JA commonly used to induce anti-herbivore defences, became more attractive to the predatory bugMacrolophus pygmaeus after exposure to far-red light (Cortéset al. , 2016). These results indicate that plants might balance their direct and indirect defences in response to signals of competition.

Macrolophus pygmaeus is a generalist predator that can feed on a broad range of herbivore species (Messelink et al. , 2011; Bouaggaet al. , 2018; Leman et al. , 2020; Kenway et al. , 2022). However, M. pygmaeus is a zoophytophagous predator, meaning that it is also able to sustain itself on plant material in the absence of arthropod prey. At higher densities, M. pygmaeus can itself cause plant damage (Castañé et al. , 2011; Sanchez et al. , 2018). The extent of plant-feeding by M. pygmaeus is dependent on the availability of arthropod prey and on the defensive status of the plants. In the absence of prey, induction of plant defences by a non-pathogenic strain of Fusarium oxysporumreduced M. pygmaeus reproduction (Eschweiler et al. , 2019). In the presence of arthropod prey, inoculation with F. oxysporum did not affect M. pygmaeus reproduction, but did lead to decreased prey abundance compared to non-inoculated plants, while prey abundance was not influenced by F. oxysporum inoculation alone (Eschweiler et al. , 2019). Together, these results indicate a shift toward more prey consumption after induction of plant defences. Reversely, reduced plant defences in low R:FR conditions might make the plant a more readily available food source for M. pygmaeus and might cause a shift toward more plant-feeding.

In this study, we further investigated the effects of changes in R:FR on the attraction and feeding behaviour of M. pygmaeus . Cortéset al. (2016) tested the effects of far-red light on the VOC emission and attractiveness of plants treated with MeJA. Although MeJA application can induce anti-herbivore responses, there are no actual herbivores present. The results reported by Eschweiler et al.(2019) highlight the importance of prey availability for the interaction between plants and M. pygmaeus . We therefore studied the effect of R:FR on the HIPV emission of plants infested with the greenhouse whitefly (Trialeurodes vaporariorum ), and how this influences the attraction of M. pygmaeus . We tested both an increase and decrease in R:FR compared to sunlight levels, as previous work indicated that both changes in R:FR could influence plant-whitefly interactions (Shibuya et al. , 2010; Meijer et al. , 2022), with possible consequences for plant-predator interactions. We also investigated how supplemental far-red light influenced the reproduction of M. pygmaeus in both the presence and absence of T. vaporariorum as prey. We expect that a reduction in R:FR influences the emission of HIPVs and increases the attraction of M. pygmaeus to whitefly-infested plants. An increase in R:FR is expected to have little or no effect, as an increase in R:FR also did not influence the performance of T. vaporariorum (Meijer et al. , 2022). We further expect that supplemental far-red light increases M. pygmaeus reproduction in both the absence and presence of prey, related to reduced plant defences in low R:FR conditions.

Materials & Methods

Plants and arthropods

Tomato plants (Solanum lycopersicum cv. Moneymaker) were used in all experiments. Greenhouse whiteflies, Trialeurodes vaporariorumWestwood (Hemiptera: Aleyrodide), were obtained from the stock colony of the Laboratory of Plant Breeding (Wageningen University, the Netherlands). The T. vaporariorum colony is maintained on tomato (Moneymaker) at 21/17˚C, 60% RH and 16h/8h L/D. The predatory bugs,Macrolophus pygmaeus (Hemiptera: Miridae), were obtained from a commercial strain of Biobest Biological Systems (Westerlo, Belgium).

Experimental design

Laboratory experiments were performed to investigate the effects of R:FR on tomato HIPV emissions and the attraction of the predatory bugM. pygmaeus . Tomato seeds were sown in rockwool blocks (7.5 x 7.5 cm) soaked in Tomato 2.0 nutrient solution (Unifarm, Wageningen, the Netherlands). Two weeks after germination, the plants were transferred to a climate-controlled growth chamber (25/18˚C day/night, 70±3% RH and 16h/8h L/D) and divided over three separate compartments with different light treatments. Each treatment consisted of 150 μmol/m²/s white light (5700K) supplemented with 60 (+FR), 30 (CL) or 0 (-FR) μmol/m²/s of far-red light (735 nm). The control light treatment (CL) was set at a R:FR of 1.2, which is equivalent to sunlight, and was reduced (+FR) or increased (-FR) to 0.6 and 8.2, respectively. Both white and far-red light were provided by Dyna LED-modules (Heliospectra, Gothenburg, Sweden). The different compartments were separated with white reflective plastic to prevent light contamination between treatments. Plants were exposed to the light treatments for one week before being exposed to T. vaporariorum whiteflies (WF). Plants were individually covered with mesh bags, tightly fastened at the base of the rockwool cube. One hundred adult whiteflies were released inside the mesh bags and left to feed freely for five days before plants were used in further experiments. Other plants remained uninfested (U) for five days under the same conditions, resulting in six treatment combinations (+FR_WF, +FR_U, CL_WF, CL_U, -FR_WF and -FR_U).

Y-tube olfactometer

The response of M. pygmaeus to plant volatiles was observed in a two-choice Y-tube olfactometer as described previously (Lins et al. , 2014). A Y-shaped Pyrex tube, formed by an entry arm (20 cm) and two side arms (13 cm, 80° angle), was positioned vertically. The two side arms were each connected to 15 L glass jars containing tomato plants as odour sources. Compressed air was passed through the jars at a regulated flow of 2.5 L/min to carry the plant volatiles into the arms of the Y-tube. Before reaching the jars, the air was filtered by passing it through active charcoal. The glass jars were isolated in light-tight cabinets, equipped with a Dyna LED module, to provide plants with their respective light treatment throughout the choice assays while preventing visual detection of the plants by the predators. A single plant was introduced in each glass jar. The rockwool base of the plants was wrapped in aluminium foil before placement into the jar and the plants were left to acclimate for 15 min before starting choice assays. Individual predators were released at the downwind arm of the Y-tube, and their choice for either odour source was recorded when they passed at least 10 cm into one of the side arms, or no choice was recorded if they did not pass this mark within 10 min after their release. Pair-wise comparisons were made between whitefly-infested plants from the three light treatments (+FR_WF, CL_WF and -FR_WF). Because M. pygmaeus is known to prefer whitefly-infested plants over undamaged plants (Ingegno et al. , 2011; Leman et al. , 2020), choice assays between uninfested and infested plants of the control-light treatment (CL_U and CL_WF) were added as a positive control. For each pair-wise comparison, the response of 10 female M. pygmaeuspredators of 7-10 days old was tested daily for a total of 13 days (130 predators per comparison). After each set of five predators, the position of the odour sources was switched between the left and right side arms to prevent positional bias. After every 10 predators, the plants were removed and the Y-tube system was flushed with clean air for 10 min before introducing new plants.

Collection of headspace VOCs

Volatiles were collected from plants of all six treatment combinations. The rockwool base of plants was wrapped in aluminium foil before plants were placed in 15 L glass jars. They were left to acclimate for 15 min before starting the headspace collection. Air was filtered through active charcoal before reaching the jars, and volatiles were collected by drawing air with a suction pump through a stainless steel cartridge containing 200 mg of Tenax TA (20/35 mesh, CAMSCO, Houston, Texas, USA) at 150 mL/min for two hours. During volatile collection, plants remained exposed to their respective light treatments. Samples were collected from at least 10 plants for each of the six treatments. Volatile samples of empty rockwool blocks soaked with Tomato 2.0 nutrient solution and wrapped in aluminium foil were also collected to correct for background odours. The Tenax cartridges with VOC samples were dry purged under a stream of helium (50 mL/min) for 15 min to remove excess moisture.

The collected volatiles were thermally released from the Tenax TA adsorbent using an Ultra 50:50 thermal desorption unit (Markes, Llantrisant, Glamorgan, UK) at 250 °C for 10 min under a 20 mL/min helium flow, while the volatiles were simultaneously re-collected in a thermally cooled universal solvent trap: Unity (Markes) at 0 °C. When desorption was completed, the volatile compounds were released from the cold trap by ballistic heating at 40 °C/s to 280 °C, which was then kept for 10 min, while all the volatiles were transferred to a ZB-5 MS analytical column (30 m x 0.25 mm ID x 1 mm F.T. with 10 m built-in guard column (Phenomenex, Torrance, CA, USA), placed inside the oven of a Thermo Trace GC Ultra (Thermo Fisher Scientific, Waltham, MA, USA) for further separation of the plant volatiles. The gas chromatograph (GC) oven temperature was initially held at 40 °C for 2 min and was then raised at 6 °C/min to a final temperature of 280 °C, where it was kept for 4 min under a constant helium flow of 1 mL/min. A Thermo Trace DSQ quadrupole mass spectrometer (Thermo Fisher Scientific) coupled to the GC was operated in an electron impact ionization (EI) mode at 70eV in a full scan with a mass range of 35–400 amu at 4.70 scans/s. The mass spectrometer (MS) transfer line and ion source were set at 275 °C and 250 °C, respectively. Automated baseline correction, peak selection (S/N > 3) and alignments of all extracted mass signals of the raw data were processed following an untargeted metabolomic workflow using MetAlign software, producing detailed information on the relative abundance of mass signals representing the available metabolites (Lommen, 2009). This is followed by reconstruction of the extracted mass features into potential compounds using the MSClust software through data reduction by means of unsupervised clustering and extraction of putative metabolite mass spectra (Tikunov et al. , 2012). Tentative identification of volatile metabolites was based on comparison of the reconstructed mass spectra with those in the NIST 2008 and Wageningen Mass Spectral Database of Natural Products MS libraries, as well as experimentally obtained linear retention indices (LRIs).

Macrolophus pygmaeus reproduction

A greenhouse experiment was conducted to study the effects of supplemental FR light on the reproduction of M. pygmaeus , in both the absence and presence of T. vaporariorum as arthropod food source. Tomato seeds were sown in rockwool blocks as described above and transported to a greenhouse compartment (22/18˚C, 70% RH and 16h/8h L/D) three weeks after germination. Plants were individually placed in mesh cages (60 x 40 x 40 cm; BugDorm, Taiwan) and divided over two tables, both illuminated with 150 μmol/m²/s broad spectrum white light provided by VYPR series LEDs (model VR-3X-BW4, Fluence, Rotterdam, the Netherlands) as supplement to natural daylight. One table was further supplemented with 60 μmol/m²/s far-red light (Philips Greenpower LEDs, Eindhoven, the Netherlands), creating a R:FR of 0.8 (+FR) compared to 1.6 in the control (CL). Ten plants were placed on either side of the greenhouse and were exposed to the light conditions for one week. After four weeks, 100 adult whiteflies of mixed age and sex were introduced to half of the cages, five in either light treatment, creating four treatments based on the supplementation of FR light and the presence of whiteflies (CL/-WF, CL/+WF, +FR/-WF and +FR/+WF). The introduction of 100 adult whiteflies occurred weekly to maintain a viable population and food source forM. pygmaeus throughout the experiment. At six weeks, 12 adultM. pygmaeus of approximately 5-10 days old (six males and six females) were introduced to each cage. After 25 days, all M. pygmaeus were collected and counted per cage. The experiment was repeated three times between April and September 2022, resulting in 15 replicates per treatment. During the second and third run, leaflets from the 6^th and 9^th leaf in the C/+WF and +FR/+WF treatments (10 plants per treatment) were collected to provide estimates of the whitefly population within the cage. Leaf discs of 2.5 cm² were excised from the three most terminal leaflets per leaf (30 leaf discs per leaf per treatment) and the number of whitefly eggs and nymphs was counted. The 9^th leaf was the youngest fully developed leaf.

Statistical analysis

Choice responses of M. pygmaeus in the two-choice Y-tube experiment were analysed with a two-sided binominal test with α = 0.05, for each pair-wise comparison separately. Data are presented as the total number of M. pygmaeus choosing either odour source in each pair-wise comparison.

The volatile emission data, expressed as peak heights, were imported into SIMCA-P 17 statistical software (Umetrics, Umea, Sweden), followed by log-transformation, mean-centering and unit-variance scaling before being subjected to multivariate data analysis. Unsupervised principal component analysis (PCA) and/or supervised partial least squares-discriminant analysis (PLS-DA), and its extension orthogonal partial least squares-discriminant analysis (OPLS-DA) were used as tools to compare and correlate treatment groups. The results of the analysis are visualized in score plots, which reveal the sample structure according to model components, and in loading plots, which display the contribution of the variables (individual VOCs) to these components as well as the relationships among the variables. R² and Q² metrics are provided for PLS-DA or OPLS-DA analysis. These metrics describe the explained variation within the data set and the predictability of the model, respectively, and were calculated based on the averages of the sevenfold cross-validation. R² and Q² values range between 0–1, and the closer these metrics are to 1, the higher the variance explained by the model and the more reliable the predictive power of the model. Significant differences in the total emission of individual VOCs between light treatments of either infested or uninfested plants were analysed using Kruskal-Wallis analysis. Pair-wise comparisons, as performed in the Y-tube choice assays, were analysed using Mann-Whitney U analyses for comparisons showing significant separation in multivariate data analysis.

Macrolophus pygmaeus reproduction, expressed as the total number of nymphs produced per six females, were analysed using a univariate general linear model (GLM) with light treatment and whitefly presence as fixed factors, and the experimental replicates as random factor. Whitefly numbers are expressed as the total number of eggs, crawlers (1^st instar nymphs) and nymphs per leaf disc. A separation between 1^st instar nymphs and older stages is made because crawlers are more easily distinguished from other nymphal stages. Data were analysed using multivariate GLM with light treatment and leaf number as fixed factors, and plant ID and the experimental replicates as random factors.

Results

Y-tube choice assays

In the positive control, M. pygmaeus significantly preferred whitefly-infested plants over uninfested plants (Fig. 1). No preference was observed when M. pygmaeus were offered the choice between whitefly-infested plants exposed to different R:FR light ratios in pair-wise comparisons. This indicates that changes in R:FR did not influence the attraction of M. pygmaeus to whitefly-infested plants.

Headspace VOC emissions

We identified 70 VOCs across the six different treatment combinations (Table S1). These compounds were detected in at least 50% of the samples from at least one of the treatment combinations. Multivariate analysis (OPLS-DA) comparing the volatile blends from uninfested plants of all three light treatments (+FR_U, CL_Uand -FR_U) did not result in a significant separation between volatile blends (permutation test: R² = 0.287, Q² = 0.024; p_cv-ANOVA = 1.000), indicating that changes in R:FR did not influence the composition of constitutive volatile blends (Fig. 2A and B). Similarly, OPLS-DA including the whitefly-infested plants of all three light treatments (+FR_WF, CL_WF and -FR_WF) also did not result in significant separation (permutation test: R² = 0.320, Q² = 0.056; p_cv-ANOVA = 0.998), indicating that HIPV emission is also not affected by changes in R:FR (Fig. 2C and D). In uninfested plants, nine VOCs were emitted in significantly different quantities by plants exposed to different R:FR ratios, all showing a gradient from highest emission in +FR to lowest in -FR, with the exception of (Z )-3-hexen-1-ol (Table S1). In whitefly-infested plants, only four VOCs showed significant differences, following the same gradient from +FR to -FR, with the exception of 2-methylbutanal-O-methyloxime (Table S1).

We also compared volatile blends pair-wise following the pair-wise comparisons performed in the Y-tube choice essays. Surprisingly, multivariate analysis (PCA) did not show a separation between the volatile blends of CL_U and CL_WF (Fig. S1). Similarly, no separation was observed between the volatile blends of CL_WF and +FR_WF, or between VOC blends of CL_WF and -FR_WF (Fig. S2). We did observe significant separation between the blends of +FR_WF and -FR_WF using PLS-DA analysis (permutation test: R² = 0.967, Q² = 0.731; p_cv-ANOVA = 0.011) (Fig. S3). Twenty-three compounds contributed most to the separation, with five VOCs showing significantly higher emission in +FR_WF and one compound showing significantly lower emission in +FR_WF (Table S2).

Macrolophus pygmaeus reproduction

The total number of M. pygmaeus nymphs collected after the 25-day period was significantly higher in the presence of whitefly prey compared to when no prey was available (Fig. 3). Furthermore, the number of nymphs was significantly higher on plants exposed to +FR compared to CL treatments. No significant interaction between the presence of prey and the light treatments was found, indicating that also in the absence of prey, supplemental FR light increased M. pygmaeusreproduction.

Trialeurodes vaporariorum density estimate

Both light treatment and leaf number had a significant effect on the number of whiteflies of all three life stages (Fig. 4). +FR significantly increased whitefly numbers compared to CL. The effect of leaf number was related to the leaf age; younger leaves (9^th leaf) contained more eggs and crawlers while older leaves (6^th leaf) had more nymphs. A significant interaction between light treatment and leaf number was only observed for nymphs (Fig. 4).

Discussion

Shade avoidance responses are part of a complex network of ecological interactions within the canopy that together shape the growth-defence dynamics in plants (de Vries et al. , 2017). The prioritization of shade avoidance responses over defensive responses leads to changes in plant-arthropod interactions that span across trophic levels (Lazzarinet al. , 2021). Previous work indicated that plants experiencing competition might trade direct defences for indirect defences by showing that plants expressing the SAS are more attractive to the predatory bugM. pygmaeus (Cortés et al. , 2016). In this paper, we further examined the effects of shade avoidance on the interaction between M. pygmaeus and tomato plants, thereby taking into account the zoophytophagous nature of this predator. We show that the volatile-mediated attraction of this predator to whitefly-infested plants is not affected by changes in R:FR, but that far-red light does influence the reproduction of M. pygmaeus and whiteflies.

Our results indicate that changes in R:FR do not strongly affect the emission of tomato volatiles and also do not influence the attraction ofM. pygmaeus to whitefly-infested plants. Cortés et al.(2016) previously reported that exposure to far-red light increased the attraction of this predator to MeJA-treated tomato plants. Whiteflies are known to induce SA-dependent defences in plants (Elzinga et al. , 2014; Xu et al. , 2019). SA- and MeJA-induced volatiles can be differentially affected by changes in R:FR, resulting in differences in predator behaviour in response to these volatiles. However, previous work showed that the attraction of predatory mites to plants infested with JA-inducing spider mites (Tetranychus urticae ) was also not affected by changes in R:FR (Meijer et al. , 2023), indicating that changes in R:FR do not influence the emission of both SA- and JA-induced HIPVs. The observed results by Cortés et al. (2016) might therefore result from the use of MeJA instead of actual herbivory. Although application of MeJA is an effective method to induce anti-herbivore responses in plants, there are differences in the responses induced by MeJA application and true herbivory (Dicke et al. 1999; Kappers et al. , 2010; Lortzing et al. , 2017; Papazian et al. , 2019), which can affect predator preference behaviour. For example, volatiles emitted by spider mite-infested plants were more attractive to a predatory mite than volatiles from JA-treated plants (Dicke et al. , 1999). VOC blends emitted by hormone-induced plants are missing the specific information pertaining to herbivore identity and density and might therefore provide a more general stress signal (Kappers et al. , 2010). Generalist predators are proposed to be more sensitive to general stress indicators compared to specific HIPVs (Silva et al. , 2021). Due to the zoophytophagous nature of M. pygmaeus , it is also possible that the presence or absence of actual herbivores can change the informational value of the volatile blends. Far-red light might alter the information provided by MeJA-induced volatile blends as it provides information on the defensive status of the plant and the accessibility of plant material, while changes in R:FR might not change the attractiveness of herbivore-infested plants as the presence of prey is the overriding element. It would be interesting to perform comparative studies between MeJA-treated and herbivore-infested plants to determine whether the presence of prey is a determining factor for the attraction of predators in plants experiencing competition.

The lack of preference between whitefly-infested plants exposed to different R:FR light ratios largely corresponds with the lack of differences in volatile blend composition, with the exception of the pair-wise comparison between +FR_WF and -FR_WF. A significant separation between the volatile blends of these treatments was observed, although M. pygmaeus did not show a preference for plants exposed to either +FR_FWor -FR_WF. Reversely, we did not find a significant separation between volatile blends of whitefly-infested or uninfested plants grown under control light conditions (CL_WF and CL_U), while M. pygmaeus showed a clear preference for CL_WF plants. A possible explanation is that compounds emitted by whitefly-infested tomato plants that are relevant for attracting M. pygmaeus were emitted at levels below the detection ability of the analytical instruments used. The volatiles detected in the headspace of tomato plants in this study primarily consisted of terpenoids (60 out of 70 compounds), which play a minor role in the attraction of mirid predators (Silva et al. , 2021). Feeding by T. vaporariorum is also known to induce the emission of the ester methyl salicylate (MeSA) by tomato plants (López et al. , 2012; Conboy et al. , 2020), which was not detected in the headspace analysis of the current study. MeSA is an important compound for the attraction of mirids (Silva et al. , 2021), and its emission can be modulated after exposure to far-red light (Cortéset al. , 2016).

The results of this study also show that exposure to supplemental far-red light increases the reproduction of M. pygmaeus in both the presence and absence of prey. These results support the hypothesis that exposure to low R:FR and the subsequent downregulation of defences increases plant quality for M. pygmaeus . Although no significant interaction between light treatment and whitefly presence was found, the effect of far-red light on predator reproduction appears to be stronger in the presence of whitefly prey, which might be related to increased prey availability. Along with the increased reproduction of M. pygmaeus , we also found increased numbers of T. vaporariorum in the +FR treatment, which corresponds with previous findings (Meijeret al. , 2022). This indicates that supplemental far-red light can stimulate M. pygmaeus population development both directly through changes in plant quality and indirectly by supporting higher herbivore densities. In previous studies, supplemental far-red light increased the population growth of spider mites (T. urticae ), allowing a more rapid population growth of the predatory mitePhytoseiulus persimilis and leading to a stronger suppression of the herbivore (Meijer et al. , 2023). Unfortunately, the time frame of the current study was too short to study population development in M. pygmaeus and to make a proper assessment on the effectiveness of whitefly control by M. pygmaeus . However, the observed results provide promising opportunities for the use of far-red light for biological control in greenhouses.

The use of far-red LEDs is an application in horticulture to manipulate plant morphology and reproductive development and thereby increase crop yield (Demotes-Mainard et al. , 2016), but at the same time, it can lead to an increased pest pressure (e.g. Meijer et al. , 2022). Cortés et al. (2016) suggested that far-red light might have a role in biological control by increasing the attraction of natural enemies. Although our current results indicate that changes in R:FR neither enhance nor interfere with the attraction of the predatory bug M. pygmaeus , supplemental far-red light might still stimulate biological control by allowing a more rapid build-up of the predator population, both in the presence and absence of pests. Macrolophus pygmaeus is often used in preventative release strategies by maintaining predator populations on banker plants, but its successful establishment depends on the species of banker plant, the crop and the availability of supplemental food (Bresch et al. , 2014; Messelinket al. , 2015; Sanchez et al. , 2021). Supplemental far-red light might contribute to increased establishment of M. pygmaeusduring preventative release.

We conclude that exposure to low R:FR conditions could indirectly lead to increased plant protection against arthropod herbivores through changes in plant defences and predator-prey dynamics, with possible applications to stimulate biological control in horticulture. However, the tritrophic interactions between plants, arthropod herbivores andM. pygmaeus are complex, and more research is required to understand how far-red light shapes the ecological interactions between trophic levels. For example, plant feeding by mirid predators can induce plant defences and increase resistance to herbivores (Pappas et al. , 2015; Bouagga et al. , 2019; Pérez-Hedo et al. , 2022; Silva et al. , 2022). Herbivores also avoid plants that have been exposed to M. pygmaeus (Zhang et al. , 2019). On the other hand, too high densities of M. pygmaeus can cause plant damage through direct feeding or the transmission of viruses (Castañé et al. , 2011; Moerkens et al. , 2016; Moerkens et al. , 2017; Sanchez et al. , 2018). As far-red light exerts strong effects on plant defensive signalling, it has the potential to influence both direct and indirect interactions between all three trophic levels. A proper understanding of how these interactions are affected by changes in R:FR is required to adequately assess the efficiency of supplemental far-red light to stimulate biological control.

Acknowledgements

This research was funded by the Dutch Research Council (NWO), project number ALWGR.2017.004 with financial contributions by Biobest Biological Systems, Enza Seeds, Florensis, Schoneveld Breeding, WPK Vegetable Plants, and Heliospectra through the NWO.

References

Aartsma, Y., Bianchi, F.J.J.A., van der Werf, W., Poelman, E.H. and Dicke, M. (2017). Herbivore-induced plant volatiles and tritrophic interactions across spatial scales. New Phytologist , 216, 1054-1063.

Ballaré, C.L. (2014). Light regulation of plant defense. Annual Review of Plant Biology , 65, 335-363.

Ballaré, C.L. and Austin, A.T. (2019). Recalculating growth and defense strategies under competition: key roles for photoreceptors and jasmonates. Journal of Experimental Botany , 70(13), 3452-3436.

Ballaré, C.L. and Pierik, R. (2017) The shade-avoidance syndrome: multiple signals and ecological consequences. Plant, Cell and Environment , 40(11), 2530-2543.

Ballaré, C.L., Scopel, A.L. and Sámchez, R.A. (1990) Far-red radiation reflected from adjacent leaves: an early signal of competition in plant canopies. Science , 247(4940), 329-332.

Bouagga, S., Urbaneja, A. and Pérez-Hedo, M. (2018). Comparative biocontrol potential of three predatory mirids when preying on sweet pepper key pests. Biological Control , 121, 168-174.

Bouagga, S., Urbaneja, A., Depalo, L., Rubio, L. Pérez-Hedo, M. (2019). Zoophytophagous predator-induced defences restrict accumulation of the tomato spotted wilt virus. Pest Management Science , 76, 561-567.

Bresch, C., Ottenwalder, l., Poncet, C. and Parolin, P. (2014). Tobacco as banker plant for Macrolophus pygmaeus to controlTrialeurodes vaporariorum in tomato crops. Universal Journal of Agricultural Research , 2(8), 297-304.

Cabedo-López, M., Cruz-Miralles, J., Vacas, S., Navarro-Llopis, V., Pérz-Hedo, M., Flors, V. and Jaques, J.A. (2019). The olfactive response of Tetranychus urticae natural enemies in citrus depend om genotype, prey presence, and their diet specialization. Journal of Pest Science , 92, 1165-1177.

Casal, J.J. (2012) Shade avoidance. The Arabidopsis book / American Society of Plant Biologists , 10.

Castañé, C., Arnó, J., Gabarra, R. and Alomar, O. (2011). Plant damage to vegetable crops by zoophytophagous mirid predators. Biological Control , 59, 22-29.

Conboy, N.J.A., McDaniel, T., George, D., Ormerod, A., Edwards, M., Donohoe, P., Gatehouse, A.M.R. and Tosh, C.R. (2020). Volatile organic compounds as insect repellents and plant elicitors: an integrated pest management (IPM) strategy for glasshouse whitefly (Trialeurodes vaporariorum ). Journal of Chemical Ecology , 46, 1090-1104.

Cortés, L.E., Weldegergis, B.T., Boccalandro, H.E., Dicke, M. and Ballaré, C.L. (2016). Trading direct for indirect defense? Phytochrome B inactivation in tomato attenuates direct anti-herbivore defenses whilst enhancing volatile-mediated attraction of predators. New Phytologist , 212, 1057-1071.

Courbier, S. and Pierik, R. (2019). Canopy light quality modulates stress responses in plants. iScience , 22, 441-452.

De Vries, J., Evers, J.B. and Poelman, E.H. (2017). Dynamic plant-plant-herbivore interactions govern plant growth-defence integration. Trends in Plant Science , 22(4), 329-337.

Demotes-Mainard, S., Péron, T., Corot, A., Bertheloot, J., le Gourrierec, J., Pelleschi-Travier, S., Crespel, L., Morel, P., Huché-Thélier, L., Boumaza, R., Vian, A., Guérin, V., Leduc, N. and Sakr, S. (2016). Plant responses to red and far-red lights, applications in horticulture. Environmental and Experimental Botany , 121, 4-21.

Dicke, M. and Baldwin, I.T. (2010). The evolutionary context for herbivore-induced plant volatiles: beyond the ‘cry for help’.Trend in Plant Science , 15(3), 167-175.

Dicke, M., Gols, R., Ludeking, D. and Posthumus, M.A. (1999), Jasmonic acid and herbivory differentially induce carnivore attracting plant volatiles in lima bean plants. Journal of Chemical Ecology , 25(8), 1907-1922.

Elzinga, D.A., de Vos, M. and Jander, G. (2014). Suppression of plant defense by a Myzus persicae (green peach aphid) salivary effector protein. Molecular Plant-Microbe Interactions , 27, 747-756.

Eschweiler, J., van Holstein-Saj, R., Kruidhof, H.M., Schouten, A. and Messelink, G.J. (2019). Tomato inoculation with a non-pathogenic strain of Fusarium oxysporum enhances pest control by changing the feeding preference of an omnivorous predator. Frontiers in Ecology and Evolution , 7, 213.

Fernández-Milmanda, G.L. and Ballaré, C.L. (2021) Shade avoidance: expanding the color and hormone palette. Trends in Plant Science , 26(5), 509-523.

Ingegno, B.L., Pansa, M.G. and Tavella, L. (2011). Plant preference in the zoophytophagous generalist predator Macrolophus pygmaeus(Heteroptera: Miridae). Biological Control , 58, 174-181.

Izaguirre, M.M., Mazza, C.A., Biondini, M., Baldwin, I.T. and Ballaré, C.L. (2006). Remote sensing of future competition: impacts on plant defenses. Proceedings of the National Academy of Sciences , 103(18), 7170-7174.

Kappers, I.F., Verstappen, F.W.A., Luckerhoff, L.L.P., Bouwmeester, H.J. and Dicke, M. (2010). Genetic variation in jasmonic acid- and spider mite-induced plant volatile emission in cucumber accessions and attraction of the predator Phytoseiulus persimilis . Journal of Chemical Ecology , 36, 500-512.

Kegge, W., Weldegergis, B.T., Soler, R., Vergeer-van Eijk, M., Dicke, M., Voesenek, L.A.C.J. and Pierik, R. (2013). Canopy light cues affect emission of constitutive and methyl jasmonate-induced volatile organic compounds in Arabidopsis thaliana . New Phytologist , 200, 861-874.

Kenway, A.H.E., El-Sheikh, W.E.A. and Mohamed, M.A. (2022). Evaluation of Chrysoperla carnea and Macrolophus pygmaeus as biological control agents of Frankliniella occidentalis on Batavia lettuce under hydroponic cultivation. Journal of Crop Protection , 11(2), 269-278.

Lazzarin, M., Meisenburg, M., Meijer, D., van Ieperen, W., Marcelis, L.F.M., Kappers, I.F., van der Krol, A.R., van Loon, J.J.A. and Dicke, M. (2021). LEDs make it resilient: effects on plant growth and defense.Trends in Plant Science , 26(5), 496-508.

Leman, A., Ingegno, B.L., Tavella, L., Janssen, A. and Messelink, G.J. (2020). The omnivorous predator Macrolophus pygmaeus , a good candidate for the control of both greenhouse whitefly and poinsettia thrips on gerbera plants. Insect Science , 27, 510-518.

Lins Jr, J.C., van Loon, J.J.A., Bueno, V.H.P., Lucas-Barbosa, D., Dicke, M. and van Lenteren, J.C. (2014). Response of the zoophytophagous predators Macrlophus pygmaeus and Nesidiocorus tenuis to volatiles of uninfested plants and to plants infested by prey or conspecifics. BioControl , 59(6), 707-718.

Lommen, A. (2009). MetAlign: interface-driven versatile metabolomics tool for hyphenated full-scan mass spectrometry data processing.Analytical Chemistry , 81, 3079-3086.

López, Y.I.A., Martínez-Gallardo, N.A., Ramírez-Romero, R., López, M.G., Sánchez-Hernández, C. and Délano-Frier, J.P. (2012). Cross-kingdom effects of plant-plant signaling via volatile organic compounds emitted by tomato (Solanum lycopsersicum ) plants infested by the greenhouse whitefly () plants infested by the greenhouse whitefly (Trialeurodes vaporariorum) . Journal of Chemical Ecology , 38, 1376-1386.

Lortzing, T., Firtzlaff, V., Nguyen, D., Rieu, I., Stelzer, S., Schad, M., Kallarackal, J. and Steppuhn, A. (2017). Transcriptomic responses ofSolanum dulcamara to natural and simulated herbivory.Molecular Ecology Resources , 17(6), e196-e211.

Meijer, D., Meisenburg, M., van Loon, J.J.A. and Dicke, M. (2022). Effects of low and high red to far-red light ratio on tomato plant morphology and performance of four arthropod herbivores. Scientia Horticulturae , 292, 110645.

Meijer, D., van der Vleut, J., Weldegergis, B.T., Costaz, T., Duarte, M.V.A., Pekas, A., van Loon, J.J.A. and Dicke, M. (2023). Effects of far-red light on tritrophic interactions between the two-spotted spider mite (Tetranychus urticae ) and the predatory mitePhytoseiulus persimilis on tomato. Submitted.

Messelink, G.J., Bloemhard, C.M.J., Hoogerbrugge, H., van Schelt, J., Ingegno, B.L. and Tavella, L. (2015). Evaluation of mirid predatory bugs and release strategies for aphid control in sweet pepper. Journal of Applied Entomology , 139(5), 333-341.

Messelink, G.J., Bloemhard, C.M.J., Kok, L. and Janssen, A. (2011). Generalist predatory bugs control aphids in sweet pepper.IOBC/wprs Bull , 68, 115-118.

Moerkens, R., Berckmoes, E., van Damme, V., Ortega-Parra, N., Hanssen, I., Wuytack, M., Wittemans, L., Casteels, H., Tirry, L., de Clerq, P. and de Vis, R. (2016). High population densities of Macrolophus pygmaeus on tomato plants can cause economic fruit damage: interaction with Pepino mosaic virus ? Pest Management Science , 76, 1350-1358.

Moerkens, R., Berckmoes, E., van Damme, V., Wittemans, L., Tirry, L., Casteels, H., de Clerq, P. and de Vis, R. (2017). Innoculative release strategies of Macrolophus pygmaeus Rambur (Hemiptera: Miridae) in tomato crops: population dynamics and dispersal. Journal of Plant Disease and Protection , 124(3), 295-303.

Moreno, J.E., Tao. Y., Chory, J. and Ballaré, C.L. (2009). Ecological modulation of plant defense via phytochrome control of jasmonate sensitivity. Proceedings of the National Academy of Sciences , 106(12), 4935-4940.

Papazian, S., Girdwood, T., Wessels, B.A., Poelman, E.H., Dicke, M., Moritz, T. and Albrectsen, B.R. (2019). Leaf metabolomic signatures induced by real and simulated herbivory in black mustard (Brassica nigra) . Metabolomics , 15(10), 1-16.

Pappas, M.L., Steppuhn, A., Geuss, D., Topalidou, N., Zografou, A., Sabelis, M.W. and Broufas, G.D. (2015). Beyond predation: the zoophytophagous predator Macrolophus pygmaeus induced tomato resistance against spider mites. PLoS One , 10(5), e0127251.

Pérez-Hedo, M., Bouagga, S., Zhang, N.X., Moerkens, R., Messelink, G., Jaques, J.A., Flors, V., Broufas, G. Urbaneja, A. and Pappas, M.L. (2022). Induction of plants defenses: the added value of zoophytophagous predators. Journal of Pest Science , 95, 1501-1517.

Sanchez, J.A., López-Gallego, E., Pérez-Marcos, M. and Perera-Fernández L. (2021). The effects of banker plants and pre-release on the establishment and pest control of Macrolophus pygmaeus in tomato greenhouses. Journal of Pest Science , 94, 297-307.

Sanchez, J.A., López-Gallego, E., Pérez-Marcos, M., Perera-Fernández, L.G. and Ramírez-Soria, M.J. (2018). How safe is it to rely onMacrolophus pygmaeus (Hemiptera: Miridae) as a biocontrol agent in tomato crops? Frontiers in Ecology and Evolution , 6, 132.

Shibuya, T., Komuro, J., Hirai, N., Sakamoto, Y., Endo, R. and Kitaya,Y. (2010). Preference of sweetpotato whitefly adults to cucumber seedlings grown under two different light sources. HortTechnology , 20(5), 873-876.

Silva, D.B., Hanel, A., Franco, F.P., de Castro Silva-Filho, M. and Bento, J.M.S. (2022). Two in one: the neotropical mirid predatorMacrolophus basicornis increased pest control by feeding on plants. Pest Management Science , 78, 3314-3323.

Silva, D.B., Urbaneja, A. and Pérez-Hedo, M. (2021). Response of mirid predators to synthetic herbivore-induced plant volatiles.Entomologia Experimentalis et Applicata , 169, 125-132.

Tikunov, Y.M., Laptenok, S., Hall, R.D., Bovy, A. and de Vos, R.C.H. (2012). MSClust: a tool for unsupervised mass sepctra extraction of chromatography-mass-spectrometry ion-wise aligned data.Metabolomics , 8, 714-718.

Van Butselaar, T. and van den Ackerveken, G. (2020) Salicylic acid steers the growth-immunity tradeoff. Trends in Plant Science , 25(6), 566-576.

Wasternack, C. and Feussner, I. (2018) The oxylipin pathways: biochemistry and function. Annual Reviews of Plant Biology , 69, 363-386.

Xu, H., Qian, L., Wang, X., Shao, R., Hong, Y., Liu, S. and Wang, X. (2019). A salivary effector enables whitefly to feed on host plants by eliciting salicylic acid-signaling pathway. PNAS , 116(2), 490-495.

Zhang, N.X., van Wieringen, D., Messelink, G.J. and Janssen, A. (2019). Herbivores avoid host plants previously exposed to their omnivorous predator Macrolophus pygmaeus . Journal of Pest Science , 92, 737-745.

Zust, T. and Agrawal, A.A. (2017). Trade-offs between plant growth and defense against insect herbivory: an emerging mechanistic synthesis.Annual Review of Plant Biology , 68, 513-534.