Advanced methods for insect nets: red-colored nets contribute to sustainable agriculture

Development of advanced pest control methods that do not rely on insecticides is an important issue for sustainable agriculture. Particularly with regards to micro pests that are not only highly resistant to various insecticides but also because we are running out of options for which insecticide to use against them, resulting in enormous economic damage worldwide. Here we report that the effectiveness of the conventional insect net can be greatly advanced by changing their color to red that helps significantly reduce pesticide use. We demonstrate the red effect using Onion thrips, Thrips tabaci a main vector of Iris Yellow Spot Virus (IYSV) and Tomato Spotted Wilt Virus (TSWV) that cause serious damage to various vegetables. New red nets succeeded in suppressing the invasion rates and damages (white spots on the leaves) in a Welsh onion greenhouse with minimum use of pesticides. We discuss how red nets are compatible with labor-saving, sustainable agriculture and the future potential of “optical pest control” based on insect color vision and its behavioral response.


Results
Behavioral responses of Thrips tabaci against the red-colored nets on Welsh onion Among the 2.0 mm mesh nets, the red-white nets reduced thrips invasion into the net by one-third compared with the white nets, which was a significant difference (Fig. 2A).In the case of the 1.0 mm mesh nets, the redwhite nets reduced thrips invasion to about one-half of the white nets, but this difference was not significant (Fig. 2A).In the 0.8 mm mesh nets, the red-black nets and the red-red nets significantly reduced thrips invasion to about 1/14 and 1/8, respectively, compared with the white nets (Fig. 2A).The difference between the two red-containing nets and the white nets was statistically significant (Fig. 2A).In addition, among the 2.0-and 1.0-mm mesh nets, the red-white nets produced fewer feeding marks than the white nets, but the difference was not significant (Fig. 2B).On the other hand, in the 0.8 mm mesh nets, the number of feeding marks was reduced to about one-ninth to one-fourth in the red-black and red-red nets compared with the white nets, and difference between red-black and white nets was statistically significant (Fig. 2B).In the analysis of the invasion rate and color of the yarn, the effects of the red yarn and the black yarn were significantly different from zero, and the absolute value of the coefficient was higher in the red yarn than in the black yarn (Table S2).
The results of the thrips invasion experiments when two types of insect nets were placed together are shown in Fig. 3A and their statistical analysis is shown in Table S3.As is clearly apparent, in all combinations of redcolored insect nets and black-white or white nets, the number of individuals entering the red-colored net was significantly lower.No significant differences were observed in any of the combinations of red-colored nets.In Asterisks indicate that the invasion rate is significantly different from 0.5 (Wald test, p < 0.05, Refer to Table S3); n.s.,Not significant.(B) Summary of results of the two-net-choice experiments (Fig. 3A, Table S3).(a) Choice probabilities are represented with arrows that point from the net with a smaller probability of choice to the net with a larger probability of choice.Statistically significant probabilities of choice rates are represented as solid lines and not significant ones (n.s.) as dashed ones.(b) A simple visual representation of the relationships "greater than" and "less than".Nets grouped by the dashed circle represent choice probabilities that are not significantly different from one another, or that their relationships "greater than" and "less than" are transitory.
In addition, nets were sorted to avoid inconsistencies in the "greater than" and "less than." Incidentally, this representation can be reworded in computer science terms 27 as follows: the graph (i.e., pair of nodes and directed edges) shown in Fig. 3B(a) is decomposed into its strongly connected components and topologically sorted as shown in Fig. 3B(b).www.nature.com/scientificreports/ the combination of black-white and white nets, the number of individuals in the white net was significantly lower.
Those results are summarized graphically in Fig. 3Ba and in a simplified form in Fig. 3Bb.From the charts in Fig. S2, the relationships "greater than" and "less than" in the choice probability of two-net-choice experiments can be summarized as RR, RW, RB < WW < BW.

Control of Thrips tabaci by covering Welsh onion fields with red-colored nets
In Trial 1, T. tabaci outbreaks in white nets and in the no net plots started on 5 July and their populations increased until 15 August (Fig. 4A).In comparison, on 15 August, the adult and larval populations in the redred net and red-white net plots were significantly reduced to about one-sixth and one-sixtieth, respectively, of Infected leaf rate (%) those in the white net plots (Fig. 4A).There was no significant difference in the adult and larval populations in red-red net and red-white net plots throughout the study period (Fig. 4A).
With respect to leaf damage rate, there were significant differences between the red-red net and the red-white nets plots compared to the white net and the no net plots (Fig. S3).As can be seen in the figure, on 2 August the damage rate in the red-red net and the red-white net plots was reduced to about 1/21 and 1/9, respectively of that in the white net plots, respectively.Furthermore, in the red-red net and red-white net plots, chlorotic spots was not observed on Welsh onion leaves until 15 August, while leaves with IYSV disease were observed from 20 July in the untreated plot and from 2 August in the white net plots (Fig. 4B).The leaf infection rate in the red-red net and red-white net plots was significantly lower than that in the white net plots on 15 August, about one-seventh and one-third lower, respectively (Fig. 4B).
In Trial 2, T. tabaci adult outbreak began on 3 July and the adult populations in full-, ceiling-, and sidescovering net plots on 1 August was significantly lower than in the no net plots (Fig. 5A).Larvae were not observed in full-covering net plots during the survey period (Fig. 5A).While there was no significant difference in the www.nature.com/scientificreports/number of larvae among ceiling-covering, sides-covering, and no net plots, the larval populations in sidescovering plots were reduced to about one-tenth of that in the no net plot (Fig. 5A).
The leaf damage rate in the full-covering net plots was significantly different from that in the no net plots throughout the trial period (Fig. S4).On the other hand, leaf damage rates in the ceiling-and sides-covering net plots remained higher than in the full-covering net plots, and significantly lower than in the no net plots on 14 August (Fig. S4).
IYSV infected leaves were not observed in the full-covering net plots (Fig. 5B).In ceiling-and sides-net plots, the leaf infection rate remained lower than in the no net plots, and on 14 August, it was significantly reduced to about one quarter and one third of the rate in the no net plots (Fig. 5B).

Discussion
Insect nets are well-known for their use as one of the physical control techniques against thrips, whiteflies, and leaf miners in the cultivation of vegetables.The effect of insect net mesh size on pest invasion rate has been investigated, but the effect of net color has not been studied at all.Our results of the two nets choice experiments (Fig. 3A) suggest that thrips visually discriminate nets containing red fibers (Fig. 3B).The compound eye of T. tabaci is thought to be comprised of three spectral types of photoreceptors: UV-sensitive, blue-sensitive, and green-sensitive 28 .Because the light environment in the laboratory does not include UV light, the photoreceptor sensitive to UV light is unlikely to be involved in the mechanism underlying the pest control effect of the red nets; blue and green-sensitive photoreceptors are likely to be involved.At wavelengths longer than 600 nm, spectral reflectance is higher in the red nets (red-red, RR; red-white, RW; and red-black, RB) than in the other nets (Fig. 1B).Therefore, the pest controlling effect could be due to the long-wavelength component of the reflected light from the net stimulating the green photoreceptor cells in thrips.The pest control effect of other reported red light and materials on thrips 11,15,29,30 could also be explained by the blue-and green-sensitive photoreceptors as well, but further analyses are needed.Ohya et al. 15 have reported on the effectiveness of red nets in controlling insects.However, they found that the infestation-suppression effect of insect nets was enhanced by the color combination of the net threads.In this study, we selected the best combination of thread colors that suppress pest infestation and statistically proved the principle that red nets inhibit pest infestation.
T. tabaci has several reproductive types, and arrhenotokous and thelytokous types have been identified in Japan 19,31 .Arrhenotokous types have developed resistance to synthetic pyrethroids 8,23,24 and the insecticide susceptibility of arrhenotokous is lower than that of thelytokous 26 .Thus, insecticide-independent pest control techniques are urgently needed.
Red insecticidal nets can be a new physical control technology.Since T. tabaci migrate onto the vegetable fields from the surrounding weeds, such as Stellaria neglecta, Stellaria media var.procera, Lamium amplexicaule L., etc. 16,32 , putting up red-colored nets at the openings of greenhouses and around the fields can also significantly reduce T. tabaci invasion.In this study, red-colored nets effectively kept the population of T. tabaci on leaves low, consistent with report in Ohya et al. 15 .In addition, we found a lower rate of leaf damage (Figs.S3 and S4) and IYSV infection (Figs.4B and 5B) due to suppression of thrips populations.The reduction in the infected leaf rate in the no net plots was likely due to the high rate of leaf damage caused by T. tabaci, and the fact that chlorotic spots are difficult to see in this situation and therefore missed.
In Trial 2, insecticide was sprayed twice, and as a result the full-covering net plots produced Welsh onion with high commercial value by keeping the density of T. tabaci infestation lower than the other test plots (Table S4).By comparison, in the no net plots the density of T. tabaci was the highest among the four test plots, suggesting that at least twice as many insecticide applications are required to produce Welsh onion with high commercial value.The ceiling-and sides-covering plots also required at least one additional spraying compared to the fullnet plots.From these results we conclude that the use of red-colored nets can reduce the number of insecticide applications in the field by 25-50%, making them a significant advancement in labor-saving and sustainable pest control technology.
The effects of light on crops have been reported to increase anthocyanin in apples 46 and promote fruit coloring in strawberries 47 .Shahak et al. 48stated that the fruit weight and quality of peaches increased when covered with red-colored nets.The effect of red-colored nets on the growth and yield of Welsh onion is still unknown and needs to be investigated.In our laboratory experiments, red-colored nets with large meshes also suppressed the T. tabaci invasion (Fig. 2A) and showed that the red thread is significantly more effective in suppressing the pest infestation than white and black nets (Table S3).It has been pointed out that insect nets deteriorate the cultivation and working conditions by increasing the temperature inside the greenhouse due to reduced breathability 9,10 .Our results indicate that this problem could be solved using red-colored nets with a larger mesh.Further field trials using red-colored nets with larger meshes should be carried out.

Laboratory tests
Insects A laboratory population of T. tabaci was established from individuals that were collected from the fields of Welsh onions located in the Kameoka City, Kyoto Prefecture, on 4 August 2015.The laboratory culture was maintained on broad beans, Vicia faba, at 25 °C under a 15L-9D photoperiod regime, as described by Sogo et al. 49 and Aizawa et al. 21.

Insect nets
In the experiments, the following six types of colored insect nets were tested: a plain-woven insect net with red polyethylene yarn in the warp and transparent yarn in the weft with (0.8, 1.0, and 2.0 mm mesh sizes; see red-white net in Fig. 1A), net with red yarn in the warp and black yarn in the weft (only 0.8 mm mesh size; see red-black net in Fig. 1A), net with red yarn in the warp and red yarn in the weft (only 0.8 mm mesh size; see red-red net in Fig. 1A), net with black yarn in the warp and transparent yarn in the weft (0.8, 1.0, and 2.0 mm mesh sizes; black-white net), net with black yarn in both warp and weft (only 0.8 mm mesh size; black and black net), and net with transparent yarn in both warp and weft (0.8, 1.0, and 2.0 mm mesh sizes; white net).Figure 1B shows the spectral reflectance of each type of net measured with a spectrometer via optical fiber connected with an integrating sphere (HS1000S, Asahi Spectra Co. Ltd., Japan).

Experiments
The following experiments were conducted in the laboratory at 25 °C under a 15L-9D photoperiod regime.The skeletons of cylinders (150 mm dia., 250 mm high) for holding up the nets were made using wire and covered with one of the six types of colored insect nets described above.For controls, the skeletons were not covered with any type of net.One seedling of Welsh onion was planted in 200-ml plastic cups (100 mm dia., 45 mm high) containing vermiculite.Fourteen-day old potted plants (with two leaves each) were placed in each of the six types of colored net and set in a plastic ventilated cage (304 mm wide, 250 mm dia., 280 mm high) for 24 h.20 female adults were then released into each ventilated cage.After another 24 h, the number of adults and the feeding marks (abrasion marks) on Welsh onion leaves were counted.The experiment was replicated 10 times.In addition, two each of the five types of 0.8 mm mesh colored insect nets (except black and black type), for a total of 10 test combinations, were selected and placed together 10 cm apart inside a plastic ventilated cage (304 mm wide, 250 mm dia., 280 mm high) with a 10 cm opening.One seedling of Welsh onion was placed inside the insect net, as in the above experiment.In the center of the plastic ventilated cage, 20 female adults were released.
The number of adults on Welsh onion leaves was counted after 24 h.This experiment was replicated 6 times.

Field trials
Two experiments were conducted in a Welsh onion field at the Kyoto Prefectural Agriculture, Forestry, and Fisheries Technology Center in Kameoka City, Kyoto Prefecture from June to August in 2016 (Trial 1) and in 2017 (Trial 2).

Trial 1
The Welsh onion field was divided into four experimental plots (each 21.6 m 2 , 5.4 m X 4 m).For each of the three test plots, the entire surface of the greenhouse framework (4.0 m width, 5.4 m depth, 2.0 m height) was covered with 0.8 mm mesh red-white, red-red, or white-white types of insect net (Fig. S1).The control plot was not covered with insect net.This experiment was repeated twice.Welsh onion plants were planted on 15 June 2016.The cultivars planted were 'Shinkujohosonegi' and 'Ryokusyu' with plant and row spacing of 14 cm and 25 cm, respectively.In all test plots, no chemical insecticides were used during the study period.Plants were monitored once every two weeks, from 5 July to 15 August 2016.We counted the number of T. tabaci individuals and leaves damaged by thrips (except on 5 July) on 10 plants in each experimental plot.The numbers of leaves with necrotic spots by IYSV were also counted.Infection of IYSV was confirmed by DAS-ELISA when chlorotic spots were observed on the Welsh onion plants in each treatment.Leaves with chlorotic spots were counted as diseased leaves.

Trial 2
As in Trial 1, the field was divided into four experimental plots (each 21.6 m 2 , 5.4 by 4 m): plot with only the framework (4.0 m width, 5.4 m depth, 2.0 m height) covered with 0.8 mm mesh red-red insecticidal netting; plot with only the ceiling covered; plot with only the sides covered, and plot not covered with any netting (Fig. S1).This experiment was replicated twice.Welsh onion plants were planted on 13 June 2017.The cultivars planted were 'Taibyosobutori' .The plant and row spacing was the same as in 2016.Due to the high densities of T. tabaci in the untreated area, all experimental plots were sprayed with spinetoram wettable powder on July 20, 2017, and with dinotefuran water-soluble powder on August 15, 2017.
The surveys were conducted as in Trial 1, at approximately two-week intervals during the period of 3 July to 14 August 2017.
All the experiments were performed in accordance with relevant institutional, national, and international guidelines and legislation.

Figure 1 .
Figure 1.(A) Red-white, red-red, and red-black nets used in the experiment.Red-white netwoven with red polyethylene thread in warp and transparent polyethylene thread in weft; Red-red net-woven with red polyethylene thread in both the warp and weft; and Red-black net: woven with red polyethylene thread in warp and black polyethylene thread in weft.(B) The reflectance spectra of nets used in the experiments.

Figure 2 .Figure 3 .
Figure 2. (A) Comparison of the pest control effect of the six types of insect nets used against Thrips tabaci female adults.There is a significant difference between samples designated with different letters (Tukey-Kramer multiple comparison test using arcsine transformed values, p < 0.05).(B) Comparison of the effectiveness of the six types of insect nets in controlling the damage caused by Thrips tabaci female adults.There is a significant difference between samples designated with different letters (Steel-Dwass multiple comparison test, p < 0.05). https://doi.org/10.1038/s41598-024-52108-1

Figure 4 .
Figure 4. (A) The effect of red insect nets on the density of Thrips tabaci per 20 Welsh onion plants in Trial 1. Columns denoted by different letters are significantly different at the 5% level by Steel-Dwass multiple comparison test performed at each survey date.(B) The effect of red insect nets on IYSV infected leaf rate in Trial 1. Columns denoted by different letters are significantly different at the 5% level by Tukey-Kramer multiple comparison test using arcsine transformed values from each survey date.

Figure 5 .
Figure 5. (A) The effect of red insect nets on the density of Thrips tabaci per 20 Welsh onion plants in Trial 2. Columns denoted by different letters are significantly different at the 5% level by Steel-Dwass multiple comparison test performed at each survey date.(B) The effect of red insect nets on IYSV infected leaf rate in Trial 2. Columns denoted by different letters are significantly different at the 5% level by Tukey-Kramer multiple comparison test using arcsine transformed values from each survey date. https://doi.org/10.1038/s41598-024-52108-1www.nature.com/scientificreports/