Evaluation of Brassicaceae Seedlings as Trap Plants for Bagrada Hilaris Burmeister in Caper Bush Cultivations

: The caper bush, Capparis spinosa (Brassicales: Capparaceae), is intensively grown on Pantelleria Island (Trapani, Sicily, Sicilian channel) where it has been granted protected geographical indication (PGI) by the EU. On this island, Bagrada hilaris , a stink bug native of Asia and Africa, is the major pest of caper crops. Recent studies have shown the attraction of B. hilaris to volatiles of brassicaceous plants at the seedling stage. The objective of this study was to evaluate three cotyledon-stage seedlings of host plants, Brassica oleracea var. botrytis (cauliﬂower), Eruca sativa (rocket) and Brassica carinata (Abyssinian cabbage), as potential trap plants for B. hilaris . The relative preferences of these species were ﬁrst evaluated in laboratory and ﬁeld experiments, carried out during summer when the level of B. hilaris infestation was the highest. Behavioral bioassays in the laboratory conditions showed that adults of B. hilaris preferred to orient toward seedlings of B. oleracea and E. sativa over B. carinata . Field experiments conﬁrmed these results. Then seedlings were tested in trap plant trials, by sowing them in artiﬁcial pots formed with aluminum trays and placing them in caper ﬁelds infested with B. hilaris . Results showed that E. sativa and B. oleracea diverted hundreds of B. hilaris individuals from the capers to these sources of attraction. Overall, these results suggest that B. oleracea and E. sativa seedlings used as lure inside traps or as trap plants may be a useful tool in the management of B. hilaris populations.


Introduction
Capparis spinosa L. (Brassicales: Capparaceae), also known as the caper bush, is a perennial shrub cultivated mainly for the flower buds known as capers, used for human consumption as spices [1,2]. Caper bush is a xerophilous plant able to develop and survive in environments with dry summer climates, typical of the islands of the Mediterranean Basin [3]. In Italy, capers are cultivated in Sicily and its minor islands where the positive use of marginal lands allows it, and has higher profit margins compared to other local crops [4]. In particular, caper cultivation on Pantelleria island has been granted protected geographical indication (Commission Regulation No. 1107/96 -EU) and produces 300-400 tons per year [5]. Caper plants are attacked by a large group of phytophagous insects including Capparimya savastanoi (Martelli) (Diptera: Tephritidae), Asphondylia gennadii (Marchal) (Diptera: Cecydomidae) and several pentatomid bugs (Heteroptera: Pentatomidae) such as Bagrada hilaris (Burmeister), Antheminia lunulata (Goeze), Eurydema ornata L., Eurydema ventralis Kol., Holcostethus punctatus (Lindberg) and Nezara viridula L. [4]. fields on the island of Pantelleria (Italy). Insects were reared in climatic cell (30 ± 2 • C, 70 ± 10% RH, photoperiod 16 L:8 D) in 47.5 × 47.5 × 47.5 cm cages (BugDorm-44545; MegaView Science Co. Ltd., Taichung, Taiwan). The colony was fed with cauliflower and cabbage plants, depending on seasonal availability. Because B. hilaris lays eggs in the soil, paper dishes (6-cm Ø) with a mixture of sand, silt, and clay (33% for each soil component) were placed in the cages as oviposition sites. Dishes were changed weekly, and those with eggs were kept in separate cages until the emergence of nymphs. The nymphs were then kept in separate cages until the final molt to adults. Newly emerging adults were used individually in laboratory experiments.

Seedlings
Seeds of B. oleracea var. botrytis (afterward named B. oleracea), E. sativa, and B. carinata were placed on cotton wool (10 g) soaked with distilled water and held in glass containers. Seeds were sown at a density of about 0.5/cm 2 . The containers were placed in an environmentally controlled growth chamber (25 ± 1 • C, 70 ± 10% RH, photoperiod 16 L: 8 D) equipped with lights with a photosynthetic flux density (PPFD) of 600 mol photons m −2 s −1 . Cotyledon-stage seedling clusters, made by 50 plants at 3-9 days from emergence, were prepared; the cotton wool substrate was wrapped in aluminum foil to avoid desiccation and then used for laboratory bioassays. The seedlings used for the experiments carried out in the field were grown in a nursery in Pantelleria, in open condition.

Laboratory Experiments
Host choice assessment of E. sativa, B. oleracea, and B carinata was carried out in the laboratory with a dual choice arena and in open vertical olfactometer bioassays. The B. hilaris choice was evaluated in two separate pairwise comparison experiments of the different species using plants at the ages of 3 and 9 days from emergence.
The arena consisted of a Plexiglas cage 50 cm × 30 cm × 30 cm high. The two side walls of the cage were made with net for ventilation. Inside the arena, two clusters of different plant species were placed 20 cm apart in the arena. Each cluster was made by n = 50 cotyledon-stage seedlings. Individuals (n = 6 per replicate) were released into the cage at 2:00 PM and the total number of insects on each seedling cluster was observed and recorded after 20 h, at 10:00 AM the next day (final choice). The position of the treatments inside the arena was alternated after each replication. After each bioassay, the Plexiglas cage was cleaned with water and dried. Observations were conducted in static air, under controlled laboratory temperature and humidity conditions (25 ± 2 • C, and 50 ± 15% RH). A total of 11 replications were carried out for each test.
Olfactometer bioassays were carried out to verify the role of volatile organic compounds (VOCs) in B. hilaris attraction to the tested cotyledon-stage seedlings. The open vertical Y-shaped olfactometer consisted of a brass rod (left and right arms 20 cm long, central arm 25 cm long, 1 cm diameter). The left and right arms were partially covered with two glass tubes (18 cm long, 5 cm diameter) terminating in hose nipples connected by Tygon tubes to a high-purity air source, and air flow was controlled with a flow-meter at a rate of 0.2 L/min. The air flowed through two glass chambers (500 mL each), which held the test stimuli (seedling clusters N = 50). Light was provided with a halogen lamp (Osram, 12 V-35 W, Münich, Germany) hanging 30 cm above the olfactometer. Experiments were carried out under controlled laboratory temperature and humidity conditions (25 ± 2 • C, and 50 ± 15% RH) starting from 10:00 AM to 15:00 PM. For each replicate, a single adult individual was gently placed at the bottom of the central arm of the olfactometer with a paintbrush and allowed 10 min to respond. Both males and females were individually used for the experiment, at a ratio of 1:1. The bugs moved from the bottom upward toward the light source and upon arriving at the Y junction, had the option to choose between the different volatile stimuli. The criterion for a response was that the test bug walked in the test arm or the control arm for at least 5 cm past the Y junction (first choice). Bugs that did not move into one of the two arms during the 10 min trial were scored as non-responders and were not included in the analysis. After 8 replications, the glass parts of the apparatus were washed with water and detergent, then wiped with acetone and the brass rod was cleaned with distilled water and acetone and baked at 200 • C for 60 min.
Insects were used separately for dual choice arena and olfactometer experiments; each individual was tested only once and then placed in rearing cages. All the insects were starved for one day before the start of the experiments.

Field Trap Bioassays
Cotyledon-stage seedlings of B. oleracea and E. sativa were tested as attractant lures in traps in a caper bush field infested by B. hilaris. A cluster of 50 cotyledon-stage seedlings (7 days old) with soaked cotton used as growing substrate were placed inside the traps. For this experiment a horizontal plastic trap (25 × 15 × 15 cm) furnished by GEA S.r.L., (Settimo Milanese Milan-Italy) was used, with a funnel-like shape on both sides to allow insect entrance. The trap used (S1) was specifically conceived and designed in consideration of B. hilaris behavior characterized by oviposition activity and mating mainly happening on the ground [5]. The traps were placed near a randomly chosen caper bush plant and partially buried in order to facilitate insect entrance and to prevent wind damage (S1). Paraffin oil was applied on the board of the inner part of the funnels to prevent the insects' escape. Four traps per each seedling species and control (empty traps with soaked cotton wool) were used. Traps were placed in a Pantelleria caper bush field of 0.5 hectares (36 • 46'15.5" N 11 • 57'43.9" E) homogeneously infested by B. hilaris, and disposed using a Latin square design. The distance between traps was approximately 8 m. The traps were inspected every 3 days for 15 days and the trapped individuals (adults and nymphs) were counted and removed. During inspections, water was provided to the seedlings to avoid their desiccation and also to control trap.

Trap Plant Evaluation
Host preference bioassays using B. oleracea, E. sativa, and B. carinata were carried out in a caper bush field in Pantelleria (36 • 46 21.7" N 11 • 57 38.1" E). The caper bush field chosen for the experiment was 40 m long and 80 m wide and presented a heavy infestation of B. hilaris. The experiment was carried out using aluminum trays (40 × 30 × 10 cm) as artificial pots, containing cotton soaked with water used as growing substrate. Each species (treatment) was sown in four trays (n = 4 for each of the three treatments) using 5 g of seeds for each tray. The seeds were sown in July 2019 in a nursery, irrigated and transferred to the field one day after emergence. Irrigation was carried out every two days. As the rows of caper bush closer to the dry stone walls surroundings the field presented the highest level of infestation, the trays were placed between the cultivating rows and distributed around the field's perimeter, alternating the treatments at a distance of 20 m from one to each other. The number of total B. hilaris individuals present on each cotyledon-stage seedling tray was counted and removed from the pot using a paint brush every 2 days until the 15th day after the seedlings' emergence. Trays were rotated clock-wise by one position after each inspection, to avoid possible position bias.

Statistical Analysis
The data obtained from the laboratory experiments in dual choice arena were analyzed using a paired t-test, while the ones obtained from the open vertical Y-shaped olfactometer bioassay were analyzed using χ 2 tests. The data obtained from the field trap bioassays were analyzed using a one-way ANOVA followed by Tukey test. Finally, the data obtained from the trap plant evaluation bioassays were analyzed using two-way ANOVA, considering the factors "treatment", "time of inspection" and their interaction, while Fisher least significant difference analysis (LSD) was used to distinguish differences among means for individuals for each inspection day. All the statistical analyses were performed using Statistica 10.0 for Windows (Statsoft, Vigonza, Padova, Italy).

Field Trap Bioassays
The results of the field trap bioassays using cotyledon-stage seedlings as lures are reported in Figure 3. Overall, the traps baited with B. oleracea and E. sativa captured more individuals than the control (F = 16.03, df = 2, p < 0.0001). More specifically, the traps baited with B. oleracea and E. sativa captured respectively a mean (± SE) of 6.29 ± 1.2 and 9.54 ± 1.65 individuals per trap per 3 days, while no captures were recorded in the control traps. However, the number of captures determined by B. oleracea and E. sativa did not differ statistically (p = 0.14; Tukey test).

Field Trap Bioassays
The results of the field trap bioassays using cotyledon-stage seedlings as lures are reported in Figure 3. Overall, the traps baited with B. oleracea and E. sativa captured more individuals than the control (F = 16.03, df = 2, p < 0.0001). More specifically, the traps baited with B. oleracea and E. sativa captured respectively a mean (± SE) of 6.29 ± 1.2 and 9.54 ± 1.65 individuals per trap per 3 days, while no captures were recorded in the control traps. However, the number of captures determined by B. oleracea and E. sativa did not differ statistically (p = 0.14; Tukey test).

Field Trap Bioassays
The results of the field trap bioassays using cotyledon-stage seedlings as lures are reported in Figure 3. Overall, the traps baited with B. oleracea and E. sativa captured more individuals than the control (F = 16.03, df = 2, p < 0.0001). More specifically, the traps baited with B. oleracea and E. sativa captured respectively a mean (± SE) of 6.29 ± 1.2 and 9.54 ± 1.65 individuals per trap per 3 days, while no captures were recorded in the control traps. However, the number of captures determined by B. oleracea and E. sativa did not differ statistically (p = 0.14; Tukey test).

Trap Plant Evaluation
The results of the trap plant evaluation are reported in Figure 4. Overall, the effect of time of inspection and treatment determined differences statistically significant (p < 0.01; ANOVA), while the interaction for the two factors was marginally not significant (p = 0.08; ANOVA). The trays with cotyledon-stage seedlings of E. sativa attracted a higher number of B. hilaris individuals (adults and nymphs) compared to B. oleracea and B. carinata at the 5th and 7th day from emergence (p < 0.05; ANOVA). However, successively the E. sativa attracted a number of individuals similar to the other species. Moreover, at the 9th, 11th, and 13th day from their emergence, the B. oleracea attracted a higher number of individuals compared to B. carinata (p < 0.05; ANOVA). Overall, the attraction of B. oleracea and E. sativa cotyledon-stage seedlings progressively decreased after the 11th day from emergence. This was in part determined by the strong feeding damage by B. hilaris that led to the death of the majority of the cotyledon-stage seedlings.

Trap Plant Evaluation
The results of the trap plant evaluation are reported in Figure 4. Overall, the effect of time of inspection and treatment determined differences statistically significant (p < 0.01; ANOVA), while the interaction for the two factors was marginally not significant (p = 0.08; ANOVA). The trays with cotyledon-stage seedlings of E. sativa attracted a higher number of B. hilaris individuals (adults and nymphs) compared to B. oleracea and B. carinata at the 5th and 7 th day from emergence (p < 0.05; ANOVA). However, successively the E. sativa attracted a number of individuals similar to the other species. Moreover, at the 9 th , 11 th , and 13 th day from their emergence, the B. oleracea attracted a higher number of individuals compared to B. carinata (p < 0.05; ANOVA). Overall, the attraction of B. oleracea and E. sativa cotyledon-stage seedlings progressively decreased after the 11 th day from emergence. This was in part determined by the strong feeding damage by B. hilaris that led to the death of the majority of the cotyledon-stage seedlings.

Discussion
The results provide evidence that cotyledon-stage seedlings of B. oleracea and E. sativa are good candidates as attractant sources for B. hilaris and may find application as a lure for traps or in trap cropping techniques.
The attraction of B. hilaris to these seedlings is mediated by the specific volatile organic compounds (VOCs) emitted from the species tested. In fact, the olfactometer bioassays firstly evidenced that the attraction to preferred hosts is mediated by olfactory cues. In particular, testing the seedlings at 3 and 9 days from emergence, B. hilaris adults oriented toward VOCs from E. sativa rather than from those of B. carinata.
Differently from the majority of phytophagous-plant interaction cases [26], the attraction in this case seems to be determined by a key odorant molecule [24,25] rather than by a blend of ubiquitous compounds as observed. It is possible that the attraction of B. hilaris adults toward E. sativa, already observed also by Joseph et al. [23], can be determined by a few key odorant molecules, which however have not yet been investigated. In the case of B. oleracea, Guarino et al. [24] found that the B. hilaris attraction to the cotyledon-stage seedling VOCs is in particular determined by an uncommon volatile

Discussion
The results provide evidence that cotyledon-stage seedlings of B. oleracea and E. sativa are good candidates as attractant sources for B. hilaris and may find application as a lure for traps or in trap cropping techniques.
The attraction of B. hilaris to these seedlings is mediated by the specific volatile organic compounds (VOCs) emitted from the species tested. In fact, the olfactometer bioassays firstly evidenced that the attraction to preferred hosts is mediated by olfactory cues. In particular, testing the seedlings at 3 and 9 days from emergence, B. hilaris adults oriented toward VOCs from E. sativa rather than from those of B. carinata.
Differently from the majority of phytophagous-plant interaction cases [26], the attraction in this case seems to be determined by a key odorant molecule [24,25] rather than by a blend of ubiquitous compounds as observed. It is possible that the attraction of B. hilaris adults toward E. sativa, already observed also by Joseph et al. [23], can be determined by a few key odorant molecules, which however have not yet been investigated. In the case of B. oleracea, Guarino et al. [24] found that the B. hilaris attraction to the cotyledon-stage seedling VOCs is in particular determined by an uncommon volatile diterpene hydrocarbon of novel identification and named brassicadiene [25], not present in the VOCs of B. carinata.
Field bioassays carried out using living seedlings inside the traps as an attractant lure not only confirmed the high attraction of B. hilaris to B. oleracea and E. sativa, but also poses the basis for the use of living germinating seeds as a new type of attractant for monitoring this species. Even if the use of vegetal material as attractant lure for trapping insects has already been exploited for other pests such as the Red Palm weevil [27,28], the use of such seedlings as an attractant is novel to our knowledge. The use of such a lure could be also of interest considering how recent experiments with pheromone-based traps for monitoring B. hilaris captured a low number of individuals [29].
Finally, the data obtained from the trap plant experiments showed that both E. sativa and B. oleracea cotyledon-stage seedlings can attract a consistent number of B. hilaris individuals to a lesser extent than B. carinata, diverting them from the surrounding caper bush plants. These results are encouraging for more in-depth exploration into the possibility of using such seedlings for trap cropping purposes to protect caper bush plants. Furthermore, in accordance with the laboratory bioassays results, B. oleracea and E. sativa elicited a different attraction on B. hilaris individuals over time, probably related to the seedling growth rate and the relative volatiles emitted. More specifically, while E. sativa attracted the highest number of individuals during the first part of the experiment, later on B. oleracea attracted a similar number of individuals. The individuals, adults of both sexes and nymphs at all stages, were pooled together to simplify the observation. It should also be pointed out that the decrease in seedling cluster attraction in the later observations could partially have been determined by the mortality caused by the large aggregations of B. hilaris individuals on the seedlings, particularly evident in the E. sativa clusters. This may require that, before E. sativa trap plants die, they must be destroyed or treated with a pesticide to avoid the B. hilaris individuals from re-infesting the caper bush plants. Otherwise, the B. oleracea seedlings showed greater attraction to B. hilaris at the 9th day after emergence.
Overall, these results suggest that the cotyledon-stage seedlings used could have potential use as trap plants to manage this pest, also encouraged by the strong needs Pantelleria caper growers have for alternative and reliable methods to control Painted bug populations. However, it is also to be stated that, even if these seedlings have showed great potential as attractant sources, more studies are needful to better understand if their application as trap plants can determine a damage reduction of B. hilaris on the caper bush, as this was not specifically assessed in this study.
In our trap plant evaluation, we decided to carry out the experiment in the summer when the B. hilaris population was at the highest level in order to attract the highest number of individuals. However, due to the high temperature, it was necessary to periodically irrigate the trays containing the young seedlings to prevent their desiccation. In order to exploit the trap cropping technique successfully, and in consideration of the well-known water shortage in the island of Pantelleria [30], it could be alternatively recommended that the brassiceous seeds are sown during rainfall periods, for example in early autumn. This tactic was also suggested by Ludwig and Kok [31], as applying the trap plants in fall could limit the size of the population entering winter diapause. However, alternatively or simultaneously the trap plants could be used in spring to catch the newly emerging colonizers before they infest the caper bushes. In consideration of the fact that B. hilaris generally overwinter in the stone walls surrounding the caper bush fields [5], this last technique could be useful if the trap plants are planted around the perimeter of the main crop in order to resemble the so-called "perimeter trap cropping method" [32]. In fact, as suggested by Shelton and Badenes-Perez [9] "the potential success of a trap cropping system depends on the interaction of the characteristics of the trap plant and its deployment with the ecology and behavior of the targeted insect pest". To our knowledge, very few cases have been reported of commercially successful trap planting applications [9]. On the other hand, it is encouraging that the use of trap plants using Brassica plants to reduce infestation was reported successfully for other pests such as the coleopteran Psylliodes chrysocephala (L.) and Ceutorhynchus pallidactylus (Marsh) [33] and for the lepidopterean Plutella xylostella (L.) [34].

Conclusions
These results have ecological value, demonstrating the attractiveness of B. hilaris to host plant cotyledon-stage seedlings other than the caper crop. The results demonstrate the ability of these seedlings to strategically divert the B. hilaris from caper bushes and be attracted to the attractant trap plants E. sativa and B. oleracea. These new findings offer the opportunity to explore these seedlings as candidates for trap planting in different times of the year, to kill the emerging individuals from diapauses or to suppress the overwintering B. hilaris. These strategies can be considered of particular interest for caper growers who are applying organic farming methods.