Can alternative host plant and prey affect phytophagy and biological control by the zoophytophagous mirid Nesidiocoris tenuis?

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1 BioControl DOI /s Can alternative host plant and prey affect phytophagy and biological control by the zoophytophagous mirid Nesidiocoris tenuis? Antonio Biondi. Lucia Zappalà. Angelo Di Mauro. Giovanna Tropea Garzia. Agatino Russo. Nicolas Desneux. Gaetano Siscaro Received: 12 April 2015 / Accepted: 5 October 2015 International Organization for Biological Control (IOBC) 2015 Abstract Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) is an important natural enemy of several key arthropod pests. However, in tomato crop this predator can cause economic damage owing to its zoophytophagous behavior. We investigated in laboratory conditions the influence of two alternative plants, Dittrichia viscosa L. (Asteraceae) and Sesamum indicum (L.) (Pedaliaceae), with or without prey, on N. tenuis damage and its biological control services on Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) eggs. Both D. viscosa and S. indicum, as companion plants in dual-choice bioassays, significantly reduced the damage of the mirid on tomato. S. indicum was more attractive than D. viscosa for feeding and oviposition and its presence did not interfere with the Handling Editor: Josep Anton Jaques Miret. A. Biondi L. Zappalà A. Di Mauro G. Tropea Garzia A. Russo G. Siscaro (&) Department of Agriculture, Food and Environment, University of Catania, via Santa Sofia 100, 95 Catania, Italy gsiscaro@unict.it A. Biondi antonio.biondi@unict.it L. Zappalà lzappala@unict.it A. Di Mauro dimauroan@gmail.com G. Tropea Garzia gtgarzia@unict.it predation on T. absoluta eggs. We also studied the potential of the three plants as preyless rearing substrate for the mirid, and only S. indicum showed to be a suitable host plant for N. tenuis development and oviposition. The potential applications of S. indicum in N. tenuis field management and mass rearing are discussed. Keywords Tuta absoluta Sesamum indicum Dittrichia viscosa Omnivory Miridae Banker plant Introduction The capacity to feed and the need to rely on multiple trophic levels for survival is the main attribute of omnivorous organisms. The multitrophic interactions A. Russo agarusso@unict.it Present Address: A. Biondi Department of Environmental Science, Policy and Management, University of California Berkeley, 1801 Walnut Street #10, Berkeley, CA 94709, USA N. Desneux French National Institute for Agricultural Research (INRA), UMR Institut Sophia Agrobiotech, Univ. Nice Sophia Antipolis, CNRS, 400 Route des Chappes, Sophia-Antipolis, France nicolas.desneux@sophia.inra.fr

2 A. Biondi et al. deriving by the presence of such organisms in simplified ecosystems, such as cultivated crops, may result in economically controversial scenarios (Rosenheim et al. 2004). When main natural enemies of agricultural pests are omnivores instead of strict carnivores, their impact on the crop plant can change as the omnivore shifts between consumption of prey and of plant. Thus, the beneficial importance of omnivores that provide biological control services while competing with pest species is counterbalanced by the potential economic damage (Coll and Guershon 2002). However, the role of generalist predators in agricultural pest biocontrol is increasingly strengthened, because there is good evidence of their efficiency as biocontrol agents (Symondson et al. 2002; Calvo et al. 2009; Ragsdale et al. 2011). Indeed, most crops are attacked by more than one species of pest, and biocontrol programs are thus increasingly based on releases of generalist predators, mainly under greenhouse conditions, against common pests such as thrips, whiteflies, spider mites, aphids and moths (Symondson et al. 2002; Zappalà et al. 2013). Generalist predators are resilient because they are able to survive and reproduce on non-pest food sources, such as pollen, nectar or plant sap of cultivated and wild plants (Coll and Guershon 2002). In this context, non-cultivated host plants can play an important role, facilitating early colonization and/or providing suitable refuges when the crop environment is not suitable, e.g. lack of prey or presence of pesticde residues (Messelink et al. 2014; Parolin et al. 2014; Saeed et al. 2015). Zoophytophagous mirid predators are currently used in several cropping systems in combination with selective pesticides (Tedeschi et al. 2001; Mollá et al. 2011; Zappalà et al. 2012a; Zhang et al. 2015) and/or with hymenopterous parasitoids that share the same host/prey species (Chailleux et al. 2013a, b; Moreno- Ripoll et al. 2014; Velasco-Hernández et al. 2015). In the Mediterranean region, the mirids Macrolophus pygmaeus Rambur, Nesidiocoris tenuis (Reuter), Dicyphus tamaninii Wagner and D. errans (Wolff) (Hemiptera: Miridae) are considered to be the main generalist predators of tomato pests (Perdikis et al. 2011; Biondi et al. 2013a; Ingegno et al. 2013). In addition, these mirids were able to successfully switch to preying an alien pest soon after its arrival in the Mediterranean, i.e., Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) (Urbaneja et al. 2009; Bompard et al. 2013; Jaworski et al. 2013; Mollá et al. 2014). In particular, N. tenuis is successfully and widely employed in biological control programs in tomato crops in Southern Europe through augmentative releases and conservative strategies (Vacante and Tropea Garzia 1994; Mollá et al. 2011; Zappalà et al. 2013; Sanchez et al. 2014), being able to effectively control whiteflies, thrips, leafminers, aphids, mites, and lepidopterans (Calvo et al. 2012; Pérez-Hedo and Urbaneja 2015). Its biocontrol activity against T. absoluta is becoming increasingly important, because the conventional control tool, i.e. insecticide applications (Desneux et al. 2010; Biondi et al. 2015), turned into a weak and unsuitable tool due to the development of T. absoluta resistance to the major compounds (Haddi et al. 2012; Campos et al. 2015; Roditakis et al. 2015) and to the effects on non-target organisms naturally and artificially present in the tomato crop (Arnó and Gabarra 2011; Biondi et al. 2013c). By contrast, N. tenuis produces by repeated feeding necrotic brown rings around stems and shoots as well as damages on flowers and fruits, thus causing economic losses (Calvo et al. 2009; Arnó et al. 2010; Castañé et al. 2011). When prey levels in the crop are low, phytophagy and/or migration may be of critical importance for the survival of N. tenuis. Under greenhouse conditions, N. tenuis populations diminish rapidly when densities of whitefly and other prey decrease (Sanchez and Lacasa 2008), and this may cause N. tenuis populations spillover into non-cultivated plants around agricultural areas (Perdikis et al. 2007). However, the knowledge of the ecological relationships between N. tenuis and its non-crop host plants is limited and deserves investigation. The rational management of this effective predator, spontaneously occurring or artificially released, is therefore crucial when including this species in the tomato cropping systems. Here we aimed at gaining knowledge on the N. tenuis sustainable management in tomato crops by using companion (banker or trap) plants, and on its mass rearing using preyless systems. We studied the potential role of two alternative host plants for N. tenuis management, the wild plant, Dittrichia (= Inula) viscosa L. (W. Greuter) (Asteraceae) native to the Mediterranean area and recently expanding worldwide, and the crop plant Sesamum indicum (L.) (Pedaliaceae) native to India and widely cultivated in tropical and sub-tropical regions. Dittrichia viscosa is reported as a natural host plant of M.

3 Can alternative host plant and prey affect phytophagy and biological control melanotoma (Perdikis et al. 2007) and N. tenuis (Cano et al. 2009), while N. tenuis is known to be a S. indicum pest in India and Japan (Ahirwar et al. 2010; Nakahishi et al. 2011). We investigated the influence of these two plant species, either with or without the availability of an alternative prey [eggs of Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae)], on (i) N. tenuis biological control services toward T. absoluta eggs and on (ii) the damage to tomato caused by its phytophagy. In addition, and in order to evaluate their potential as preyless rearing substrate for the mirid, the N. tenuis reproduction outcomes on these plant/prey systems were also assessed. Materials and methods Biological materials Experimental plants were grown under greenhouse conditions into 0.2 l plastic pots with pre-mixed potting soil until they reached a height of 30 cm. Plants were watered and fertilized following routine practices and pesticide applications were strictly avoided. Seedlings of S. indicum (cv. T-85 Humera) and of tomato (Solanum lycopersicum L.) of the cv. Shiren were obtained from commercially available seeds, while for growing the D. viscosa plants we collected seeds from wild plants of the suburbs of Catania (Italy). To overcome the cold requirements of the seeds they were maintained at 6 C for at least two weeks before sowing. The laboratory colonies of T. absoluta and N. tenuis were started from specimens collected from various commercial tomato crops located in South Eastern Sicily (Ragusa and Siracusa provinces, Italy) during a survey of resident hymenoptera parasitoids attacking T. absoluta (Zappalà et al. 2012b). Field collected specimens were re-introduced in the colonies twice a year. The mirid colony was maintained in rearing cages ( cm), made of tubular structure in PVC fixed on a containment tray at the base and entirely covered with a nylon fine mesh, under laboratory conditions (26 ± 1 C, 50 ± 10 % RH and 16:8 L:D). Tomato plants were used as feeding and oviposition substrate. Ephestia kuehniella eggs (Entofood, Koppert, The Netherlands) were provided on plant leaves as factitious prey ad libitum (De Puysseleyr et al. 2013; Mollá et al. 2014). Twelve tomato plants, 50 cm high, in 0.2 l pots were placed inside the cages with 100 newly molted (B48 h) adults (sex ratio 1:1). The adults were removed after 72 h. The plants bearing the emerged nymphs were cut after additional ten days and isolated with E. kuehniella eggs in transparent ventilated plastic boxes ( cm) until adult emergence. The T. absoluta colony was maintained in the laboratory using caged tomato plants following the methodology described by Biondi et al. (2013b). Dual-choice bioassays Dual-choice trials were conducted inside cages ( cm), made out of tubular structure in PVC entirely covered with a nylon fine mesh, under standard laboratory conditions (26 ± 1 C, 50 ± 10 % RH and 16:8 L:D). To test the influence of the presence of alternative prey and/or plant on predator preference, ten different combinations were tested, and one target tomato plant (T), either infested with T. absoluta eggs (t) or not, was paired with a companion plant. The latter consisted either in one of the two alternative host plants, i.e. S. indicum (S) or D. viscosa (D) provided or not with eggs of E. kuehniella (e) as factitious prey, or in another tomato plant without the factitious prey. Tomato plants bearing T. absoluta eggs were obtained placing eight potted plants (six treatments? two control plants) inside the same kind of cages described above and exposing them to eight T. absoluta adults (sex ratio 1:1) per plant during 48 h. Subsequently, the number of T. absoluta eggs per plant was scored and those exceeding 20 were removed using a fine paint brush. The tomato plants were then moved into the cages used for the dual choice trials, while the two control plants were moved into a different cage and kept under the same environmental conditions. This was aimed at providing specific control data on egg hatchability. Twenty E. kuehniella eggs per plant were placed on the alternative plants (S. indicum or D. viscosa) distributing them on the younger leaves randomly. For each choice test, two plants (a target tomato plant and a companion plant) were exposed for 48 h to a pair (one female?one male) of naïve mirid adults. Two to three days old N. tenuis females were used in the bioassays to ensure they were ready to oviposit, since their pre-oviposition period is lasting two days (De Puysseleyr et al. 2013). The two experimental plants

4 A. Biondi et al. were placed in two opposite corners of the cage, ensuring that they were not in contact. Predators were released placing the open vial in the cage centre. After the exposure to the mirids, each plant was individually isolated into clean cages. Each plant/prey combination trial was replicated eight times. The assessment of the plant/prey combination attractiveness toward N. tenuis adults was done recording the mirid phytophagy on tomato, its oviposition preference and the T. absoluta biocontrol services provided. Feeding damage on tomato plants was recorded 72 h after insect removal by counting the total number of necrotic rings on the main stem, shoots, leaf and foliar petioles (Castañé et al. 2011). Assessment of the oviposition preference was estimated by counting the number of nymphs emerged from each plant 8, 11, 14 and 19 days after the exposure to the N. tenuis females. Nesidiocoris tenuis predatory activity was evaluated in terms of T. absoluta egg hatching percentage reduction observed 1, 3, 5, 7 and 9 day(s) after the removal of the N. tenuis specimens from the cage. Tuta absoluta egg hatching generally occurs between four and six days from the exposure to the adults (Lee et al. 2014), thus the eggs that did not hatch after nine days from the exposure to N. tenuis adults were considered preyed on. The hatching reduction (HR) was calculated comparing the number of emerged larvae to the initial number of eggs (20), corrected by the mean egg mortality recorded on the control plants using the formula: HR = [1 - (H x /H c )], where H x is the number of T. absoluta eggs hatched in the treatment x and H c the average number of T. absoluta eggs hatched in the control plants. Host plant suitability The suitability of tomato, D. viscosa and S. indicum plants for the development of N. tenuis nymphs and the survival and oviposition of adults, without additional food or water source, was assessed in the laboratory (26 ± 1 C, 50 ± 10 % RH and 16:8 L:D). We used an experimental arena made up of two superposed plastic cups that contained the excised upper plant part (about 17 cm long) of the tested plants (see Biondi et al for a detailed description of the arena). Thirty newly emerged nymphs (B12 h) per each tested plant were individually isolated into the arena and plant material was renewed every three days. The survival of each nymph was observed daily until adulthood. Then, the specimens were separated per sex and, due to the emergence of only ten females from the thirty isolated nymphs, the juvenile development duration of ten randomly selected males and of the ten females was considered for data analysis. Newly molted adults (B12 h) originated from the rearing were placed in pairs (one male and one female) into the experimental arena described above with the host plant. Twenty pairs per each host plant were tested. Survived females were transferred daily into a new arena with new plant material until adult death. Dead N. tenuis males were replaced promptly. Arenas containing exposed plant material were maintained for daily checks of nymph emergences (fertility) during the seven days following adult removal. Data analysis Datasets were first tested for normality and homogeneity of variance using Kolmogorov Smirnov D test and Cochran s test respectively, and transformed pffiffiffiffiffiffiffiffiffiffiffi pffiffi ( x þ 1 and arcsin ð x Þ for numerical and percentage values, respectively) if needed. Untransformed data are presented in tables and figures. General Linear Model was used to analyze the effects of three fixed factors on the number of necrotic rings and of nymphs emerged from the exposed plants. In the model the following fixed factors were included: companion plant species (tomato, S. indicum or D. viscosa), presence of T. absoluta eggs on the target tomato plant and presence of alternative prey on the companion plant and all their two-way interactions. Moreover, for each dual choice test, the percentages of nymphs emerged with the tomato and the companion plants were analyzed as follows: the null hypothesis that the N. tenuis oviposition choice was not affected by the treatment (the plant and prey combination), i.e. 50:50, was subjected to a v 2 goodness of fit test. The null hypothesis was rejected at P B For analyzing the effects on the T. absoluta egg HR, the percentage of preyed T. absoluta eggs was subjected to General Linear Model using companion plant species, presence of alternative prey and their interaction as fixed factors. One-way ANOVAs were carried out to test the effect of the treatment (i.e. plants/prey combination) on the number of necrotic rings and of emerged

5 Can alternative host plant and prey affect phytophagy and biological control nymphs, and percentages of preyed T. absoluta eggs in the dual choice bioassays, as well as data on juvenile survival and development, adult fertility and longevity recorded on the three tested rearing plants. Finally, means were compared using an LSD test (P B 0.05). Results Dual-choice bioassays Phytophagy on tomato The number of necrotic rings on tomato plants was significantly affected by the treatment, i.e. the plant species/prey combinations (F 9,70 = , P \ 0.001) (Fig. 1). The presence of T. absoluta eggs significantly affected the number of necrotic rings on target tomato plants (F 1,71 = , P \ 0.001), namely increased the tomato damage when the companion plant was tomato (Tt vs. T) or D. viscosa (Tt vs. De and Tt vs. D) (Fig. 1). While the number of necrotic rings was significantly lower in the treatments where uninfested target tomato plants were paired with D. viscosa (T vs. D) and especially in all the combinations with S. indicum (Tt vs. S, T vs. S and T vs. Se). The companion plant species (F 2,71 = , P \ 0.001) significantly influenced the number of rings on target tomato plants. Higher levels of phytophagy on target tomato plants were recorded when the companion plant was tomato, while the lowest when target tomato plants were paired with S. indicum. Moreover, the interaction of presence of T. absoluta eggs and the companion plant species factor was also significant (F 2,71 = 7.276, P = 0.001), indicating that the effects of the T. absoluta egg presence varied among the three companion plants, i.e. was determinant when D. viscosa was the companion plant (see Tt vs. De and T vs. De) and was not in the case of S. indicum (Fig. 1). The presence of alternative prey (F 1,71 = 0.090, P = 0.765) and its interactions with the other two factors [presence of T. absoluta eggs 9 presence of alternative prey (F 1,71 = 1.674, P = 0.200); companion plant species 9 presence of alternative prey (F 1,71 = 0.090, P = 0.765)] did not affect significantly the N. tenuis phytophagy, although the number of necrotic rings on target tomato plants in the treatments T vs. Se and T vs. De were among the lowest (Fig. 1). Reproductive outputs on tomato and companion plants The number of nymphs emerged on the target tomato plants, with or without T. absoluta eggs, when coupled with the companion plants varied among treatments significantly (F 9,70 = 7.079, P \ 0.001) (Fig. 2). The companion plant species (F 2,71 = 8.992, P \ 0.001) N of necrotic rings / tomato plant a a a ab bc bc c cd cd d Tt vs. T Tt vs. De T vs. T Tt vs. D T vs. D Tt vs. S T vs. De T vs. S Tt vs. Se T vs. Se Plants and prey combination N of nymphs / tomato plant 10 a ab ab ab 8 ab 6 4 bc 2 c cd d d 0 Tt vs. D Tt vs. De T vs. T Tt vs. T Tt vs. S Tt vs. Se T vs. De T vs. D T vs. S T vs. Se Plants and prey combination T= S. lycopersicum, S= S. indicum, D= D. viscosa, t= T. absoluta eggs, e= E. kuehniella eggs T= S. lycopersicum, S= S. indicum, D= D. viscosa, t= T. absoluta eggs, e= E. kuehniella eggs Fig. 1 Nesidiocoris tenuis phytophagy on target tomato plants expressed as mean numbers (±SE) of necrotic rings per plant. Data refer to each dual choice bioassay in which one tomato plant, with or without T. absoluta eggs, and one companion plant (either tomato, Sesamum indicum or Dittrichia viscosa), with or without E. kuehniella eggs, were exposed to one N. tenuis pair for 48 h. Columns bearing different letters are significantly different (ANOVA followed by LSD test at P B 0.05) Fig. 2 Nesidiocoris tenuis progeny on target tomato plants expressed as mean numbers (±SE) of emerged nymphs per plant. Data refer to each dual choice bioassay in which one tomato plant, with or without T. absoluta eggs, and one companion plant (either tomato, Sesamum indicum or Dittrichia viscosa), with or without E. kuehniella eggs, were exposed to one N. tenuis pair for 48 h. Columns bearing different letters are significantly different (ANOVA followed by LSD test at P B 0.05)

6 A. Biondi et al. significantly affected the number of nymphs emerged from target tomato plants. In those target tomato plants paired with S. indicum (e.g. in T vs. Se and T vs. S), the progeny outputs were significantly lower than in those paired with D. viscosa (e.g. T vs. De, Tt vs. D and Tt vs. De) and with another tomato plant (e.g. Tt vs. T and T vs. T). The number of emerged nymphs was higher in presence of T. absoluta eggs (F 1,71 = , P \ 0.001) irrespectively of the companion plant species. The interaction presence of T. absoluta eggs 9 companion plant species had no significant effect (F 2,71 = 1.083, P = 0.344). The presence of alternative prey (F 1,71 = 0.088, P = 0.768) and its interaction with the presence of T. absoluta eggs (F 1,71 = 0.400, P = 0.529) and with the companion plant species (F 1,71 = 0.097, P = 0.756) were not significant. Indeed, the number of nymphs emerged from the target tomato plants paired with alternative plants provided with E. kuehniella eggs (i.e. in T vs. De, Tt vs. De, T vs. Se and Tt vs. Se) strongly varied from 0.0 to 6.57 per plant. Nesidiocoris tenuis reproduced significantly more progeny on S. indicum, either with or without E. kuehniella eggs, than on target tomato plants, either with or without T. absoluta eggs (Fig. 3). When D. viscosa was offered together with target tomato plants, the predators produced a significantly higher number of nymphs on the alternative plant in the treatments T vs. D, T vs. De, Tt vs. De, but not when the prey was present only on tomato, i.e. Tt vs. D. No significant differences in the number of emerged nymphs on the tomato plants were found in the trials using only tomato plants with or without T. absoluta eggs (Fig. 3). Biocontrol of Tuta absoluta eggs on tomato Tuta absoluta egg hatching in control plants was always [90 % and to averaged to 96 ± 2.67 %. The percentages of preyed T. absoluta eggs on target tomato plants varied significantly among treatments (F 4,35 = 5.757, P = 0.001). Significantly higher predation rates were found in target tomato plants paired with D. viscosa plants with or without E. kuehniella eggs (Fig. 4). As a consequence, only the companion plant species (F 2,35 = , P \ 0.001) significantly affected this parameter, while there were no significant effects of the presence of alternative Fig. 3 Nesidiocoris tenuis progeny produced in each dual choice bioassay exposing target tomato plants, with or without T. absoluta eggs, and companion plants (either tomato, Sesamum indicum or Dittrichia viscosa), with or without E. kuehniella eggs, to one N. tenuis pair for 48 h. Data are expressed as mean percentages (±SE) on the total of nymphs emerged in the dual choice bioassay, results of the v 2 goodness of fit tests are also presented % of T. absoluta egg hatching reduction a ab Tt vs. D Tt vs. De Tt vs. Se Tt vs. T Tt vs. S Plants and prey combination T= S. lycopersicum, S= S. indicum, D= D. viscosa, t= T. absoluta eggs, e= E. kuehniella eggs Fig. 4 Nesidiocoris tenuis predation on Tuta absoluta eggs on target tomato plants, expressed as mean percentages (±SE) of egg hatching reduction following exposure of tomato plants and companion plants (either tomato, Sesamum indicum or Dittrichia viscosa), with or without E. kuehniella eggs, to one N. tenuis pair for 48 h. Columns bearing different letters are significantly different (ANOVA followed by LSD test at P B 0.05) prey (F 1,35 = 0.032, P = 0.859) and its interaction with the companion plant species (F 1,35 = 2.030, P = 0.163). bc c c

7 Can alternative host plant and prey affect phytophagy and biological control Host plant suitability In absence of prey, N. tenuis nymphs did not successfully develop into adults feeding either on tomato or on D. viscosa plants, while % of the nymphs reared on S. indicum plants reached the adulthood (F 2,87 = , P \ 0.001) (Table 1). On S. indicum the duration of the postembryonic development was significantly different between the sexes (F 1,18 = 9.04, P = 0.008) (Table 1). The lifetime reproductive outputs of N. tenuis females was higher on S. indicum than on D. viscosa and tomato (F 2,57 = , P \ 0.001) (Table 1). This was a combined effect of significantly lower adult longevity (females: F 2,57 = , P \ 0.001; males: F 2,57 = , P \ 0.001) and daily fertility (F 2,57 = 29.07, P \ 0.001) on D. viscosa and tomato (Table 1). Discussion The potential role of alternative plants for N. tenuis field management was assessed by means of dualchoice laboratory tests. In this scenario, i.e. when the predators had to choose between a target tomato plant (infested or not with T. absoluta eggs) and a companion one (with or without the alternative prey), S. indicum was significantly more attractive than tomato and D. viscosa for the mirid oviposition (Fig. 3). Moreover, the feeding damage was much lower or even null on the target tomato plants paired with S. indicum (Fig. 1) suggesting that this alternative plant was a good food plant source for N. tenuis, thus disrupting the mirid phytophagy on the cultivated plant effectively. However, the T. absoluta egg predation on target tomato plants paired with S. indicum (with or without alternative prey) did not decrease, thus proving that the predator actively foraged in the tomato seedling. Nevertheless, we did not quantify the feeding activity on this alternative plant and, to confirm this hypothesis, specific feeding behavioral bioassays should be conducted to quantify properly the N. tenuis feeding activity on S. indicum plants. Another factor that influenced the N. tenuis damage on the target tomato plants was the presence of T. absoluta eggs that increased the mirid damage. However, this was mediated by the companion plant species. In fact, when the companion plants were S. indicum or D. viscosa (either with or without the alternative prey) the damage on tomato was significantly lower than in the control, i.e. in T vs. T. In a whitefly-infested tomato greenhouse crop, Sanchez (2009) showed that the tomato plant damage is directly related to N. tenuis presence and inversely to prey abundance, suggesting that the amount of N. tenuis plant feeding decreases when feeding on prey increases. This kind of behavior fits the Switching hypothesis for which omnivorous predator plant feeding rate increases as prey feeding rate decreases (Gillespie and McGregor 2000). By contrast, in our laboratory experiments more rings were recorded in T. Table 1 Mean (± SE) percentages of N. tenuis nymph survival, development time, adult longevity and lifetime and daily fertility on the three tested plants in the absence of prey Tomato Dittrichia viscosa Sesamum indicum Nymphal survival 0.00 ± 0.00 % b 0.00 ± 0.00 % b ± 0.03 % a Nymph development time (days) Females 14.2 ± 0.65 A Males 11.4 ± 0.67 B Lifetime fertility (nymphs per female) 2.03 ± 0.62 b 2.45 ± 0.74 b ± 5.76 a Daily fertility (nymphs per female per 0.22 ± 0.06 b 0.21 ± 0.06 b 1.43 ± 0.21 a day) Longevity (days) Females 8.92 ± 0.62 b ± 0.77 b ± 3.39 a Males 9.81 ± 0.68 b 7.00 ± 0.43 b ± 4.24 a Values followed by different lower case letters within the same row, for nymphal survival, fertility and longevity, and in the same column by different upper case letters, for juvenile development time on S. indicum, are significantly different (ANOVA, LSD test at P \ 0.05)

8 A. Biondi et al. absoluta-egg infested tomato plants than in the healthy ones, thus supporting the Facilitation hypothesis in which plants and prey feeding (rings and T. absoluta egg presence) seem to be positively correlated. In this case, the mirid would need to acquire key components (nutrients or water) from the plant in order to optimize the prey digestion and/or assimilation (Gillespie and McGregor 2000). Besides, another explanation for the divergent results between our study and the study by Sanchez (2009) could be that whiteflies represent a more suitable prey than T. absoluta eggs for N. tenuis and that, in this case, the predator needs to compensate its food intake with plant sap. In additions, the study by Sanchez (2009) was conducted on a multigenerational field context, in which the peak of mirid populations followed the prey decline and matched the higher tomato damage levels. However, here we studied the mirid feeding only over a single pest generation and the long term demographical consequences were not taken into account. When the zoophytophagous insect has the possibility of choosing the host plant, it seems to follow the alimentary resources at the lowest trophic level at which it feeds (the favorite plants), and not resources of the upper trophic level, the prey. Moreover, our results suggest that the addition of the alternative prey (E. kuehniella eggs) does not significantly reduce N. tenuis damage on tomato, i.e. no significant differences were recorded between the treatments Tt vs. D and Tt vs. De, T vs. D and T vs. De,Tt vs. S and Tt vs. Se, T vs. S and T vs. Se (Fig. 2). Altogether these results hint the hypothesis about the prevalent N. tenuis phytophagous habit. Indeed, in a recent paper, Pérez-Hedo et al. (2015) proved the induction of a physiological response in tomato plants by N. tenuis feeding activity which results in attraction of a whitefly parasitoid and antixenosis to the whitefly itself. This kind of adaptative trophic interaction, i.e. negative toward a competitor pest and positive for a natural enemy of the competitor pest, is typical of strictly phytophagous mirid species such as for example Lygus spp. (Rodriguez-Saona et al. 2002). In realistic agricultural foodweb contexts, where multiple prey and natural enemy species can coexist, food supplements are thought to enhance pest control (Vandekerkhove and De Clercq 2010; Messelink et al. 2014). In the specific case of N. tenuis on tomato, E. kuehniella eggs or E. kuehniella eggs plus sugars have been proposed for enhancement of the mirid establishment after inoculative and augmentative releases (Calvo et al. 2012; Urbaneja-Bernat et al. 2015). By contrast, even if E. kuehniella eggs represent an excellent food source for this predator (De Puysseleyr et al. 2013; Mollá et al. 2014), in our study the biological control toward T. absoluta eggs by N. tenuis adults was not affected by the presence of this factitious prey, either when placed on S. indicum and on D. viscosa plants, i.e. no significant differences were recorded between the treatments Tt vs. D and Tt vs. De and between Tt vs. S and Tt vs. Se. This was not the case for M. pygmaeus that decreased the amount of aphid consumption when it was supplied with additional food, pepper flowers, on three different host plants and at several prey densities (Lykouressis et al. 2014). Besides, the predation on T. absoluta eggs was overall significantly higher on those tomato plants paired with D. viscosa, either with or without E. kuehniella eggs, suggesting that the predator spent more time on the tomato plants and that D. viscosa is not preferred for the predator foraging activity when tomato with T. absoluta eggs is available. The N. tenuis preference for S. indicum was further confirmed by the mirid reproductive outputs that in the choice tests were always higher on S. indicum than on tomato, irrespective of the presence of the primary or of the alternative prey. Moreover, in absence of prey and in constant optimal environmental conditions, only S. indicum showed to be a suitable host plant for N. tenuis population development, while neonate mirids transferred to D. viscosa and tomato did not complete their development into adults successfully. Adults developed from juveniles feeding on tomato and E. kuehniella eggs (i.e. those predators coming from our rearing and thus used for the bioassays) successfully switched to preyless S. indicum plants, but not to tomato and D. viscosa ones. Indeed, both longevity and reproduction were extremely low on these two latter plants. The data on unsuccessful development on tomato agree with those by Urbaneja et al. (2005) and by Nakaishi et al. (2011), while the suitability of S. indicum as host plant without prey was earlier reported by the latter authors. Nevertheless, our results show higher adult longevity and reproductive outputs than those found by Nakaishi et al. (2011) at very similar laboratory conditions. Altogether, these data show a great potential for S. indicum to be used as rearing substrate for N. tenuis, and, in contrast to those previously found, without any

9 Can alternative host plant and prey affect phytophagy and biological control additional food source, either proteinic or sugary. Previous studies evaluating different cultivated Solanaceous plants indicated that a diet deriving exclusively from vegetable substrate is insufficient for the development of this mirid, Besides, the availability of a protein fraction, deriving from predation, was considered essential to enable the postembryonic development and the reproduction of N. tenuis (Urbaneja et al. 2005). A plantless rearing methodology was recently experimented successfully, but in that case, the mirid had to feed on a nutritionally superior food, such as E. kuehniella eggs (De Puysseleyr et al. 2013). Although our results must be confirmed in a multigenerational situation, we have reasons to believe that S. indicum provides the protein fraction needed for the development and biological performances of N. tenuis. This hypotesis is corroborated by the nutritional composition of S. indicum plants that is very rich in protein, carbohydrates, and minerals (Mbaebie et al. 2010). Field studies are still needed to understand how increasing the tomato crop biodiversity with S. indicum would affect the functioning of this agroecosystem, both in the protected and in the open field context. The potential of S. indicum as biocontrol plant to ameliorate the activity of N. tenuis is greater than a typical banker plant that needs to be prior infested with an alternative prey (Messelink et al. 2014; Parolin et al. 2014). In fact, in this case the S. indicum plant itself would provide both an alternative food and oviposition site and shelter. This could favour the first establishment in the cultivated sites in absence of crop and/or of prey. In China S. indicum has been reported as important reservoir of natural enemies, both predator and parasitoids, of rice pests, and it is considered as highly eligible for applications of ecological engineering strategies (Zhu et al. 2013, 2014). At the same time, this plant can serve as trap plant if planted near the crop plants in the last portion of the tomato growing cycle, i.e. when the N. tenuis population levels are high and when most of the damage occurs (Arnó et al. 2010). Trap plants are usually used to attract, divert, intercept, and/or retain targeted insects reducing the damage to the main crop and can be a main component of pest suppression strategies, being sprayed with insecticides when pests reach high densities on these plants (Parolin et al. 2014; Lin et al. 2015). Dittrichia viscosa is well-known as a common overwintering host plant for various mirid species in the Mediterranean (Perdikis et al. 2007; Cano et al. 2009), but our results clearly show that this plant alone is inadequate as satisfactory feeding substrate for the predator. As a consequence, it is very likely that in the N. tenuis case the ecological role of D. viscosa in maintaining the mirid populations is necessarily linked with the co-presence of prey. Dittrichia viscosa infestations by specialized aphids [(e.g., Capitophorus inulae (Passerini) (Hemiptera: Aphididae)] are common, and this plant was proven as a suitable substrate for oviposition and development of M. melanotoma in the presence and absence of that prey (Perdikis et al. 2007). However, a decline in the population of M. pygmaeus was recorded when feeding solely on the plant and poor performance was observed in the presence also of prey (Lykouressis et al. 2008). Considering that N. tenuis has been observed on this plant in the Mediterranean region, it is worthwhile investigating whether D. viscosa presence near tomato can favour the N. tenuis early appearance and conservation. The use of zoophytophagous predators for pest control in vegetable crops might be thought as hazardous, but recent evidences support the theory that mirid predators are able to keep the populations of very injurious arthropods much below the economic damage thresholds. Thus, their conservation and in some cases augmentation is warranted in sustainable cropping systems. However, among mirid predators of the Mediterranean basin, N. tenuis is at the same time one of the most effective biological control agent and a potential dangerous pest of tomato particularly in mild climate regions. In this context, the research of new alternative non-crop plant and/or the combination of alternative plant and prey for this very important omnivorous plant-feeding predator is crucial. Our results suggest that S. indicum without additional prey has a great potential to be used for N. tenuis in augmentative and conservative biological control strategies in tomato crops, without affecting the biological control provided against T. absoluta eggs, and for its preyless mass-rearing. Moreover, the S. indicum attractivity toward N. tenuis, and the great mirid plasticity in exploiting food sources, can be manipulated synergistically to attract/maintain predator populations in proximity of the crops, either after artificial releases for reducing the dispersal on non-

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