Combination of UV and green light synergistically enhances the attractiveness of light to green stink bugs Nezara spp

The southern green stink bug Nezara viridula and its congener N. antennata are important agricultural pests worldwide. These species show positive phototaxis and their compound eyes have high sensitivity to UV and green lights. The attractiveness of monochromatic UV, green lights and combined UV and green light to stink bugs was investigated under field conditions. The number of stink bugs caught increased with the number of UV LEDs, but very few bugs were caught using green light, irrespective of the number of LEDs. However, the most stink bugs were caught when both colors were combined. These results indicate that monochromatic green light is less attractive to Nezara bugs, but when mixed with UV light, it synergistically enhances the attractiveness of UV light. This finding contributes to the construction of reliable and highly specific light traps to monitor Nezara bugs. The addition of green light hardly affected the attractiveness of the UV light to other insects, such as Anomala beetles, which are often caught in light traps. We conclude that the spectral composition of light that is attractive to nocturnal insects depends on the species, hence it is possible to make ecologically friendly light traps that are target specific.


Scientific Reports
| (2022) 12:12279 | https://doi.org/10.1038/s41598-022-16295-z www.nature.com/scientificreports/ In this study, we evaluated the attractiveness of mono-and multichromatic UV and green lights under field conditions to develop an effective light trap for Nezara bugs. We also evaluated the attractiveness of mono-and multichromatic lights to other stink bugs and beetles that are often caught in light traps. Our study reports novel finding that green light synergistically enhanced the attractiveness of UV light to some stink bugs, including Nezara spp, and provides insights into developing environmentally friendly species-specific traps.

Results
Attractiveness of light sources to Nezara species. Nezara spp. showed a light intensity-dependent reaction to UV light but were less attracted to green light. They also showed a reaction to the synergistic combination of these two colors. The number of Nezara bugs caught tended to increase as the number of UV-LEDs increased (Fig. 1A,E). Traps with 42 and 84 UV-LEDs caught significantly more Nezara bugs than those with 12 UV-LEDs (Shirley-Williams test; p < 0.05). Few bugs were caught in the green light traps, irrespective of the number of LEDs and there were no significant differences between the green lights (Shirley-Williams test; p > 0.05), while many stink bugs were caught in UV light traps (Fig. 1B,F). Traps with alternating 42 UV-and 42 green-LEDs caught significantly more stink bugs than traps with only 84 UV-or 84 green-LEDs (Wilcoxon signed-rank test with Bonferroni correction; p < 0.05) (Fig. 1C,G). A similar result was obtained for N. viridula in another field experiment conducted in Yamaguchi (Fig. 1D).
Attractiveness of combined light sources to other stink bug species. More than five times as many Piezodorus hybneri (Wilcoxon signed-rank test with Bonferroni correction; p < 0.05) and eight times as many Glaucias subpunctatus (but not significantly more, p > 0.05) were caught in the combined color light trap Figure 1. Attractiveness of different light sources to Nezara viridula (top) and N. antennata (bottom). The graphs on the left present the attractiveness of UV light at different intensities, the middle graphs present the attractiveness of green light at different intensities, and the graphs on the right present the attractiveness of the combined UV and green light. The number after UV or G indicates the number of LEDs used for the light source. Boxplots represent the median value (horizontal line), mean value (cross mark), interquartile range (boxed area), maximum and minimum values (vertical bar), and outlier value (circle). Asterisks indicate significant differences (p < 0.05) between 12 UV-LEDs in graphs A and E (Shirley-Williams test), and between 84 UV-LEDs in graph F (Wilcoxon signed-rank test). Different letters in the same graph indicate significant differences between light sources (Wilcoxon signed-rank test with Bonferroni correction; p < 0.05 in graphs C, D, and G). NS not significant (p > 0.05). The data in graphs A and C were obtained from field experiments conducted in Okinawa, data in graphs B, D, F, and G were collected in Yamaguchi, and data in graph E were collected in Niigata. Due to time constraints, the insects captured in traps were counted every 7 days in Okinawa, and every 3-4 days in Niigata and Yamaguchi. Attractiveness of combined light sources to beetle species. The difference in attractiveness between the three light sources was almost the same in the three Anomala beetles ( Fig. 3A-C). Only a few Anomala beetles were caught in the green light traps, while significantly more beetles were caught in the UV and combined color light traps (Wilcoxon signed-rank test with Bonferroni correction; p < 0.05). Holotrichia parallela was caught in traps with monochromatic green and UV lights (Fig. 3D). Most H. parallela were caught in the trap with combined UV and green light, but there was no significant difference in the numbers caught among the light traps (Friedman test: χ 2 = 0.1739, df = 2, p = 0.9167).

Discussion
Our field bioassays with different light traps demonstrated that monochromatic green light was less attractive for Nezara bugs, but when combined with UV light, it synergistically enhanced the attractiveness of UV light. There have been several reports of the attractiveness of monochromatic light to insects, but only a few have investigated the attractiveness of combinations of light colors. Kirkpatrick et al. 20 compared the attractiveness of green and UV light alone and in combination to stored product insects but did not find any significant differences between either of the monochromatic light and the combined color light. The greenhouse whitefly T. vaporariorum is attracted to monochromatic green light, which is increased when combined with UV light 19 . Similarly, the Asian citrus psyllid, Diaphorina citri, is strongly attracted to UV combined with yellow or green light 21 . In contrast, blue light suppresses the attractiveness of green light to whitefly 22 . Thus, the responses to combinations of colored lights differ depending on the species or wavelength composition. To the best of our knowledge, this is the first report to demonstrate that the combination of different wavelengths of light synergistically enhanced trap catches www.nature.com/scientificreports/ of nocturnal insects. In addition, these results were obtained from field evaluations, where combinations of UV and green light can be readily used for practical applications. The compound eyes of insects have several photoreceptor cells with different spectral sensitivities, and each photoreceptor cell perceives a specific range of wavelengths. In N. viridula, there seem to be three types of photoreceptor cells that are sensitive to UV, blue, and green light 17 ; thus, the UV and green lights used in our experiments could be perceived as different stimuli. Why are Nezara bugs strongly attracted to a combination of UV and green light? One possible explanation is that stimuli with two different wavelengths that cause different output behaviors might improve the capture efficiency. Different wavelengths are known to induce different "wavelength-specific behaviors" in some diurnal hemipteran insects. UV radiation elicits behavior involved in flight initiation, migration, and dispersal [22][23][24] , while green-yellow light induces settling on host plants [22][23][24][25] . T. vaporariorum prefers illumination of these two different lights (UV and green) together 19 . Even in nocturnal stink bugs, different behaviors might be elicited by light of different wavelengths. Stink bugs often pass next to a UV light source and fewer bugs land on or hit a UV light source 16 . Although UV alone is highly attractive to Nezara bugs, it seems that the accuracy of orientation to the stimulus is not necessarily high. If green light elicits orientation or landing in these bugs, which often feed on young seeds of green plants such as soybeans and rice, the green light could improve the orientation accuracy. In addition, green light may enhance downward flight and promote landing, which lead the bugs into the trap chamber placed under the light source. The wavelengthspecific behavior of nocturnal insects has not been well documented. The validity of this hypothesis could be clarified by observing the orientation position for each wavelength light or the process of capture in the trap in more detail.
Another possibility is that the simultaneous input of the two wavelengths made the light source of the trap more attractive to the insects as an orientation target. Previous studies have shown that vision plays an important role in the stable orientation of nocturnal insects 26 . Flying insects in particular need to accurately orient to objects such as flowers, leaves, and branches, either to land or to avoid during flight. To do this, they must be www.nature.com/scientificreports/ able to distinguish between the sky and objects by using the faint light of the moon or night sky. Wehner 27 suggested that insect sensitivity to both UV and blue-green wavelengths is related to wide-field motion detection, that is, orientation and navigation using the contrast between the UV-rich sky and the UV-unreflective earth. Similarly, Möller 28 suggested that insects can use UV-green or UV-blue contrasts to distinguish between celestial and terrestrial objects. In this study, the UV and green LEDs were placed approximately several centimeters apart, so that the insects could perceive the light source as a space where the sky and objects had greater contrast and were easier to distinguish from the surroundings. It is possible that the insects were strongly attracted to the light source with two wavelengths as a more reliable and stable flight orientation. Although green light enhanced the attractiveness of UV light to stink bugs, the degree of attractiveness differed greatly, even in the stink bug subfamily (Pentatomidae: Pentatominae). The color combination was synergistic and strong for P. hybneri and G. subpunctatus as well as Nezara species, but additive and weak for H. halys and P. stali. This indicated that UV light dependency, and the roles or the degree of contribution of other light differed among stink bug species. The addition of green light hardly affected the attractiveness of the UV light to Anomala beetles, whereas monochromatic green light was attractive and additively enhanced the attractiveness of the UV light to H. parallela. These results suggest that strong attractiveness to a combination of UV and green light is not common in nocturnal insects and depends on the species. In addition, the compound eyes of Anomala beetles have photoreceptors that are sensitive to green, blue, and UV and show similar spectral sensitivity curves 29 to N. viridula 17 . Therefore, the reactivity to combined light does not correspond to the known spectral sensitivity curves of these insects.
Overall, these results can contribute to making reliable, highly accurate monitoring traps for Nezara stink bugs. The results also show that the light intensity or the number of LEDs can be reduced while maintaining a threshold of attractiveness, saving power and costs. In addition, since many nocturnal insects are attracted to artificial light 30,31 , ecological disturbance caused by light has become a serious problem [32][33][34] . Even in light traps, many non-target species are caught and killed, which complicates sorting. Therefore, it is preferable to use a light source that is target specific. Taken together, our findings can contribute to the development of both economically and environmentally friendly monitoring light traps for stink bug species, including Nezara bugs.

LED traps. We used a commercially available portable light trap (Eco-chu trap, Konan Shisetsu Kanri,
Okinawa, Japan) to modify the light source. A prototype trap equipped with 12 UV-LED bulbs was developed to catch the green chafer Anomala albopilosa (Hope) 35 , but it was not sufficiently attractive to stink bugs. Light sources with different numbers of LEDs, from 12 to 84, were used. Either or both bullet-type UV-LED bulbs (NS395L-ERLO; 395 nm, 20 mA, Nitride Semiconductors, Tokushima, Japan) and green LED bulbs (NEPG510S; 525 nm, 20 mA, Nichia, Tokushima, Japan) were used. LEDs were arranged vertically on a stainless-steel cylinder (4.8 cm in diameter, 20 cm in height). Light sources with 12 LEDs were arranged in six rows around the circumference. Each row was arranged as two LEDs at 7.8 cm intervals. Adjacent LEDs were arranged in a left-handed spiral (depression angle of 53°, at approximately 2.5 cm intervals). Light sources with 21 LEDs were arranged in eight rows around the circumference. Each row was arranged as two or three LEDs at 7.2 cm intervals. Adjacent LEDs were arranged in a left-handed spiral (depression angle of 63°, at approximately 2.0 cm intervals). Light sources with 42 LEDs were arranged in eight rows around the circumference. Each row was arranged as five or six LEDs at 3.6 cm intervals. Adjacent LEDs were arranged in a left-handed spiral (elevation angle 63°, at approximately 2.0 cm intervals). Light sources with 84 LEDs were arranged in eight rows around the circumference. Each row was arranged as 10 or 11 LEDs at 1.8 cm intervals. Adjacent LEDs were arranged in a left-handed spiral (elevation angle 63°, at approximately 2.0 cm interval). When both UV and green LEDs were used, both LEDs were arranged alternately in a row (Fig. 4). The cylinder with the LEDs was covered with a transparent acrylic cylinder (9.8 cm in diameter, 20 cm in height).
The light source was mounted on a funnel (31 cm in diameter, 24 cm in height), and the lower part of the light source was approximately 100 cm above the ground. A cylindrical chamber (23 cm in diameter, 20 cm in height) was placed under the funnel so that insects that were attracted to the light fell into the funnel and were trapped. The legs of the trap were anchored to the ground using steel stakes. A dimethyl-dichloro-vinyl-phosphate (DDVP) plate containing 10.7 g dichlorvos (Bapona, Earth Chemical, Tokyo, Japan) was placed inside the chamber to kill the insects. The lights were turned on at 18:00 and turned off at 6:00 the next day. The power for the lights was supplied by rechargeable car batteries (N-40B19R/SB; DC 12 V, 28 Ah, Panasonic, Osaka, Japan) or domestic electricity power supplies (AC100V).
Emission spectra of combined UV and green light. The spectral intensity of combined UV and green light was measured using a high-speed spectrometer (HSU-100S, Asahi Spectra, Tokyo, Japan) in a dark room. An attached sensor fiber was placed 50 cm in front of the light source. The measurement was performed five times, the light source was rotated for each measurement to minimize the angle effect, and the average was used as a representative value. The UV-and green-LED emission spectra showed single peaks at wavelengths of 400 and 526 nm, respectively (Fig. 5). Calculated light intensities of UV (350-450 nm) and green (451-600 nm) regions were 2.12 × 10 17 and 2.03 × 10 17 photons m −2 s −1 , respectively; that is, the light intensities of UV-and green-LEDs were almost equal.     green-LEDs were used as light sources. Each of the three LED traps was spaced more than 30 m apart and placed randomly around the soybean field or grassland. Although insects other than Nezara bugs (mainly coleopteran species) were captured in the light traps, for soybean pests, the funnel-type light traps are intended for monitoring large coleopteran and heteropteran insects (> 1 cm). Therefore, we targeted and counted insects that meet these conditions. Statistical analysis was performed on species with a total capture number of more than 20 individuals in the three traps. The species were as follows: in addition to Nezara bugs, heteropteran bugs, Piezodorus hybneri (Gmelin), Glaucias subpunctatus (Walker), Halyomorpha halys (Stål), and Plautia stali Scott, as well as coleopteran beetles, Anomala albopilosa (Hope), A. cuprea Hope, A. rufocuprea Motschulsky, and Holotrichia parallela Motschulsky. The insects captured in traps were counted for each species every 7 days at Okinawa (total 12 replicates) and every 3-4 days at Yamaguchi (total 14 replicates). The traps were randomly repositioned every week. The raw capture data for each trap are listed in Supplementary Table S3.
Data analysis. In Experiment 1, the effect of UV light intensities for trap catches were analyzed using a nonparametric one-tailed Shirley-Williams test under an assumption that higher light intensity attracts larger amounts of insects. In Experiment 2, the effect of green light intensities for trap catches were analyzed using the Shirley-Williams test. Subsequently, the attractiveness of each green light was compared to that of UV light using Wilcoxon matched pairs signed-rank test. In Experiment 3, the effect of light sources for trap catches was analyzed using the Friedman test, followed by the Wilcoxon signed-rank test, with Bonferroni correction for multiple comparisons. Statistical analyses were performed using R version 4.2.0 (R Core Team, 2022).

Data availability
All data that support the findings of this study are provide in the manuscript and supplementary file or are available from the corresponding author on reasonable request.