Herbivore-Induced Rice Volatiles Attract and Affect the Predation Ability of the Wolf Spiders, Pirata subpiraticus and Pardosa pseudoannulata

Simple Summary The spiders, Pirata subpiraticus Bösenberg et Strand (Araneae: Lycosidae) and Pardosa pseudoannulata Bösenberg et Strand (Araneae: Lycosidae) are important natural enemies of many rice pests. Herbivore-induced plant volatiles can attract natural enemies to pest locations and are becoming important in integrated pest management. This study assessed the effects of herbivore-induced rice volatiles on the selection behavior, predation ability and field attraction of two species of spiders. The selection frequency of spiders for methyl salicylate, linalool, and 2-heptanone were significantly greater than the blank group. Methyl salicylate can shorten the predatory latency of male P. pseudoannulata and can also trap more P. pseudoannulata in the field. Linalool may also shorten the predatory latency of male P. subpiraticus and increase the daily predation capacity of female P. pseudoannulata. In summary, herbivore-induced rice volatiles attract P. pseudoannulata and P. subpiraticus, and potentially increase their pest control capability. These results provide support for the practical use of herbivore-induced rice volatiles to attract and retain spiders in rice fields. Abstract Spiders are important natural enemies of rice pests. Studying the effects of herbivore-induced rice volatiles on spider attraction and predation ability may lead to safer methods for pest prevention and control. In this study, four-arm olfactometer, predation ability experiment, and field trapping experiment were used to evaluate the effects of herbivore-induced rice volatiles on Pirata subpiraticus Bösenberg et Strand (Araneae: Lycosidae) and Pardosa pseudoannulata Bösenberg et Strand (Araneae: Lycosidae). The 0.5 μg/μL linalool concentration was attractive, and also shortened the predation latency in male P. subpiraticus and female P. pseudoannulata. The 0.5 μg/μL linalool concentration increased the daily predation capacity of female P. pseudoannulata. Male P. pseudoannulata were attracted to 1.0 g/L methyl salicylate, which also shortened their predation latency. In field experiments, methyl salicylate and linalool were effective for trapping spiders. Herbivore-induced rice volatiles attract rice field spiders and affect their predatory ability. These results suggest that herbivore-induced rice volatiles can be used to attract spiders and provide improved control of rice pests.


Introduction
Rice is an important food crop and rice yield is always a high priority [1]. However, pests such as Nilaparvata lugens Stal (Homoptera: Delphacidae), Sogatella furcifera Horváth (Homoptera: Delphacidae), Chilo suppressalis Walker (Lepidoptera: Pyralidae), and Cnaphalocrocis medinalis Guenee (Lepidoptera: Pyralidae) can reduce rice production [2]. Pesticide application is the main approach for managing rice pests. Although chemical pesticides are effective, their widespread use has resulted in an increase in the occurrence of 3R P. subpiraticus and P. pseudoannulata were collected from a rice field in Yanlou Town, Huaxi District, Guiyang, China (106 • 6 24" E, 26 • 3 19" N). Spiders of various growth stages were collected and placed in individual plastic test tubes (12 cm × 3.5 cm diam). To maintain humidity, a water-soaked sponge was placed at the bottom of each test tube, and the top of the tube was sealed with a cotton ball. Spiders were raised in a clear artificial climate box at 25 • C ± 1 • C, 75% ± 5% relative humidity, and a 14:10 h (L:D) photoperiod. The spiders were fed Musca domestica adults twice a week (2-3 M. domestica adults each time) until they were adults.

Experimental Treatments 2.3.1. Spider Selection Behavior Experiments in Response to HIRVs
A four-arm olfactometer was used following the procedure of Vet et al. [36,37] (Figure 1). The test cavity of the olfactometer was 15 cm in diameter and composed of transparent Plexiglas. Four odor areas and centers were drawn on the surface of the olfactometer's test cavity. The purpose of the area boundaries was to determine spider location. Silicon tubes were used to link the odor source bottle (or control bottle), gas cleaning bottle, activated carbon filter bottle, flow meter, and atmosphere collector to the olfactometer. A 20 W fluorescent bulb was placed 30 cm above the olfactometer. A gas flow meter was used to regulate the airflow of the four arms at 400 mL/min. The indoor test temperature was 25 • C ± 3 • C.

Spider's Predatory Ability Experiments
We used P. subpiraticus and P. pseudoannulata as experimental subjects to dete if HIRVs could affect the predatory ability of rice field spiders. The test spiders starved for seven days before predation testing, since spiders can ingest and store su food. This was done to ensure that the spiders would be able to hunt during the e ments. During the experiment, we placed filter paper containing 3 μL of the volati the plastic tube with spiders for 30 min, then placed Drosophila melanogaster (prey developed, and with similar body sizes) into the plastic tube with spiders. Prelim experiments show that spiders of different sizes and sexes have different predatory ties. Therefore, females of P. pseudoannulata placed 40 preys, and other spiders pla preys. Twenty female or male adults of each spider species were tested for each and the predatory latency (the period between the preys were placed and the spide cessfully finished the attack), as well as daily predation was recorded. The spiders were starved for 48 h before being tested (water was added without food). Liquid paraffin was used as the control and to dilute the volatile standards. Before starting the volatile test, we first observed the spider's selection behavior of the four arms of the olfactometer for placed liquid paraffin. During the test, one of the four arms was defined as the control arm, while the other three were defined as the treatment arms. The filter paper (4 cm diam) was sprayed with 20 µL quantities of different concentrations of the same volatiles (20 µL liquid paraffin was used as a control) and placed in the olfactometer's four odor source bottles. Second, we pumped the air for 5 min to fill the pipe with the odors then used a funnel to introduce the test spiders into the test chamber of the olfactometer where they were observed for 5 min. We recorded spider movements in each odor area and in the central area. If the spider entered the arm of a certain odor area and remained there for 2 min, the arm volatile was deemed the spider's last choice. The spider was deemed unresponsive if it did not make a choice within 5 min after entering the test cavity. When the spider made a final choice in one of the arms, the remaining time (5 min minus the time spent making the final choice) was added to that arm. Each time, one spider was tested, and each volatile was tested on 30 female or male adults of each species. We changed the filter paper in the bottle after five tests were completed, cleaned the olfactometer with 100% ethanol, and dried it with a hair drier. We linked the olfactometer to each odor source bottle and the control bottle after each cleaning.

Spider's Predatory Ability Experiments
We used P. subpiraticus and P. pseudoannulata as experimental subjects to determine if HIRVs could affect the predatory ability of rice field spiders. The test spiders were starved for seven days before predation testing, since spiders can ingest and store surplus food. This was done to ensure that the spiders would be able to hunt during the experiments. During the experiment, we placed filter paper containing 3 µL of the volatile into the plastic tube with spiders for 30 min, then placed Drosophila melanogaster (prey, well-developed, and with similar body sizes) into the plastic tube with spiders. Preliminary experiments show that spiders of different sizes and sexes have different predatory abilities. Therefore, females of P. pseudoannulata placed 40 preys, and other spiders placed 30 preys. Twenty female or male adults of each spider species were tested for each HIRV, and the predatory latency (the period between the preys were placed and the spider successfully finished the attack), as well as daily predation was recorded.

Field Trapping Experiments
Xixiu District, Anshun, Guizhou Province, China (106 • 9 19 E, 26 • 9 38 N) was selected as the site for the field experiment. The rice was in the filling stage. We selected volatiles such as methyl salicylate, linalool, and 2-heptanone, which were all attractive to spiders in experimental treatments. The attractant was formulated in an 8:1 proportion of volatiles to liquid paraffin. A 225 mL plastic cup served as the trap. The 2 mL attractant centrifuge tube was connected to the inside of the plastic cup with small holes drilled at the bottom and liquid paraffin was the control. We set traps between rice plants at the base of every 16 clusters of rice, 10 traps for each volatile. We recorded the species and the number of spiders in the traps after 7 days. In each HIRVs field experiment, there were three replications with a plot spacing of at least 10 m to minimize inter-plot interference.

Statistical Analysis
Experiment data were analyzed using IBM SPSS Statistics 21.0. The selection frequency of spiders between different concentrations of volatiles was tested using a goodness-of-fit χ 2 test. The observed behavioral responses were compared to expected frequencies assuming a random distribution of spiders to volatiles (three concentration odor sources and one control). The stay (retention) time of spiders in different areas was compared using one-way analysis of variance (ANOVA). The attack latency and daily predation of spiders between different treatments were assessed using one-way ANOVA in the predation experiment. In the field experiment, one-way ANOVA was used to compare spider trapping rates between the different treatments.

Spider Selection Behavior in Response to HIRVs
Liquid paraffin was used as the control and to dilute the volatile standards. The P. pseudoannulata and P. subpiraticus selection frequencies showed no significant difference between liquid paraffin in the four arms of the olfactometer (Table 1). Note: CK indicates liquid paraffin, MeSA = methyl salicylate, HE = 2-heptanone, CH = cis-3-hexen-1-ol, LI = linalool; "NS" = no significant difference; "*" denotes a significant difference at the p < 0.05 level; "**" denotes a significant difference at the p < 0.01.
We studied the duration that spiders remained in different treatments to see if HIRVs affected their selection behavior. Male P. pseudoannulata remained significantly longer in the presence of 1.0 µg/µL methyl salicylate and 1.0 µg/µL linalool than in other concentrations of the same volatiles or the control treatment. Female P. pseudoannulata remained significantly longer in the presence of 0.5 µg/µL linalool than in the presence of other concentrations of the same volatile or the control treatment. Male P. subpiraticus remained Insects 2022, 13, 90 6 of 13 longer with 1.0 µg/µL 2-heptanone and 0.5 µg/µL linalool than with other volatile concentrations or the control treatment (Table 2).

Daily Predation Capacity and Predatory Latency of P. subpiraticus
There was no difference in the predatory latency of female P. subpiraticus between treatments. The predatory latency of male P. subpiraticus was not significantly different between control and 2-heptanone (0.5 µg/µL), but linalool (0.5 µg/µL) treatment was significantly shorter than that of control and 2-heptanone (0.5 µg/µL). The daily predation capacity of female and male P. subpiraticus was not significantly different between the treatments (Figure 3).

Daily Predation Capacity and Predatory Latency of P. pseudoannulata
The predatory latency of female P. pseudoannulata was not significantly different between the treatments. The predatory latency of male P. pseudoannulata was not significantly different between the control, linalool (1.0 μg/μL), and linalool (0.5 μg/μL). However, the 1.0 ug/uL methyl salicylate treatment predatory latency was significantly shorter than that of the control, linalool (1.0 μg/μL), and linalool (0.5 μg/μL). The daily predation of male P. pseudoannulata was not significantly different between the treatments. The daily predation capacity of female P. pseudoannulata was not significantly different between the control, linalool (1.0 μg/μL), and methyl salicylate (1.0 μg/μL), but the linalool (0.5 μg/μL) treatment was significantly greater than the control treatment ( Figure 4).

Daily Predation Capacity and Predatory Latency of P. pseudoannulata
The predatory latency of female P. pseudoannulata was not significantly different between the treatments. The predatory latency of male P. pseudoannulata was not significantly different between the control, linalool (1.0 µg/µL), and linalool (0.5 µg/µL). However, the 1.0 µg/µL methyl salicylate treatment predatory latency was significantly shorter than that of the control, linalool (1.0 µg/µL), and linalool (0.5 µg/µL). The daily predation of male P. pseudoannulata was not significantly different between the treatments. The daily predation capacity of female P. pseudoannulata was not significantly different between the control, linalool (1.0 µg/µL), and methyl salicylate (1.0 µg/µL), but the linalool (0.5 µg/µL) treatment was significantly greater than the control treatment ( Figure 4).

Field Trapping
The HIRVs were attractive to spiders, and the average catch of traps with HIRVs was higher than that of control traps. In particular, the traps containing methyl salicylate had higher catches than other traps. In the HIRVs traps, the proportions of P. pseudoannulata in the total spider number were methyl salicylate (72%), linalool (62.5%), and 2-heptanone (16.67%). The proportions of P. subpiraticus in the total spider number were 2-heptanone (16.67%), methyl salicylate (12.5%), and linalool (4%) ( Figure 5).

Field Trapping
The HIRVs were attractive to spiders, and the average catch of traps with HIRVs was higher than that of control traps. In particular, the traps containing methyl salicylate had higher catches than other traps. In the HIRVs traps, the proportions of P. pseudoannulata in the total spider number were methyl salicylate (72%), linalool (62.5%), and 2-heptanone (16.67%). The proportions of P. subpiraticus in the total spider number were 2-heptanone (16.67%), methyl salicylate (12.5%), and linalool (4%) ( Figure 5).

Field Trapping
The HIRVs were attractive to spiders, and the average catch of traps with HIRVs was higher than that of control traps. In particular, the traps containing methyl salicylate had higher catches than other traps. In the HIRVs traps, the proportions of P. pseudoannulata in the total spider number were methyl salicylate (72%), linalool (62.5%), and 2-heptanone (16.67%). The proportions of P. subpiraticus in the total spider number were 2-heptanone (16.67%), methyl salicylate (12.5%), and linalool (4%) ( Figure 5).

Discussion
Spiders are among the most abundant predators in rice fields. P. subpiraticus and P. pseudoannulata are the predominant species of paddy field spiders. They have large populations and strong predatory ability [22,25]. HIPVs are specific volatile substances that plants release when they are damaged by pest feeding [38,39]. HIPVs are an important component of biological control and attract natural enemies to pest feeding locations [6,8]. The olfactometer is a useful tool that can simulate the emission of volatiles in a field environment and is useful for real-time observation of spider behavior [40]. Olfactometers have been widely used to determine the relationship between insect selection behavior and volatiles such as Chilo suppressalis Walker (Lepidoptera: Pyralidae) [41], Cotesia urabae Austin et Allen (Hymenoptera: Braconidae) [40], Harmonia axyridis Pallas (Coleoptera: Coccinellidae) [42], Coccinella septempunctata L. (Coleoptera: Coccinellidae) [43], Diaeretiella rapae McIntosh (Hymenoptera: Braconidae) [43], Adoxophyes honmai Yasuda (Lepidoptera: Tortricidae) [44], and Diaphania indica Saunders (Lepidoptera: Crambidae) [45]. We used a four-arm olfactometer to observe the selection behaviors of P. subpiraticus and P. pseudoannulata on four kinds of HIRVs. We obtained results for P. subpiraticus and P. pseudoannulata selection frequency and stay time using the four-arm olfactometer. These experiments have practical implications, because the selection behaviors of P. subpiraticus and P. pseudoannulata were strongly correlated with the types and concentrations of the attractants.
Our research aimed to determine the influence of HIRVs on spider predation responses. Therefore, the attack latency and daily predation capacity were selected to evaluate the predation ability of P. subpiraticus and P. pseudoannulata. Drosophila melanogaster Meigen (Diptera: Drosophilidae) is a commonly used model organism in the laboratory. D. melanogaster is often used as a predator laboratory for alternative prey since it is easy to cultivate, rich in nutrients, and capable of flight [57][58][59][60]. Spiders generally only consume living prey and the lively D. melanogaster stimulate predation responses in spiders. In this study, D. melanogaster was used as an alternative prey for P. subpiraticus and P. pseudoannulata. The results showed that 1.0 µg/µL methyl salicylate significantly shortened the attack latency of male P. pseudoannulata and 0.5 µg/µL linalool significantly shortened the attack latency of P. subpiraticus and enhanced the daily predation capacity of female P. pseudoannulata. Lycosid spiders mainly live at the base of rice plants where high densities of brown planthoppers often occur. The brown planthopper is the target prey of many Lycosidae spiders [26]. After the brown planthoppers feed on rice, the rice releases methyl salicylate, linalool, and other volatiles [12,61]. These volatiles stimulate the olfactory selective response in spiders, shortening predation latency and increasing predation. This may be because spiders are attracted to the odor, which may affect their feeding response.
Other studies on spider predation have shown that selected chemicals can shorten spider predation latency or enhance their predatory function. Suitable physiologically active plant substances can significantly enhance the predatory function of P. pseudoannulata on N. lugens [62,63]. Optimal low-dose pesticides, which shorten the subduing and feeding times of spiders on prey, will enhance insect control efficiency and the instant attack rate on the prey of P. pseudoannulata, P. subpiraticus, P. astrigera L. Koch (Araneae: Lycosidae), and Coleosoma octomaculatum Bösenberg et Strand (Araneae: Theridiidae) [64][65][66][67].
Spider density will affect the management of pest populations, and increasing the number of spider predators will aid in insect pest control. There are many examples of using pheromones to attract and retain natural enemies. Simpson et al. used methyl salicylate as an attractant for crops and found that parasitic wasps remain longer in these attractant crops [68]. Jaworski et al. also proved that placing methyl salicylate attractants in orchards increased the effect of predators in controlling pests compared to untreated orchards [69]. Our field experiments showed that the spider trapping rate, using methyl salicylate and linalool as attractants, was greater than that of the control treatment. Zhu and Park also found that traps baited with methyl salicylate were highly attractive to adult C. septempunctata in field tests [46]. These results show that natural enemies might use methyl salicylate as an olfactory signal to locate prey. The trapped P. pseudoannulata dominated, which showed that HIPVs not only attracts P. pseudoannulata in the laboratory but also attracts P. pseudoannulata in the field. The P. subpiraticus was trapped in smaller numbers, which may be because P. subpiraticus mainly wanders on the water surface and the base of rice plants bordering the water surface, while the traps used for this experiment were placed above the water surface. The other spiders trapped were mainly Salticidae and Clubionidae spiders, which are good at jumping, and they may have accidentally fallen into the trap. According to a preliminary field study, P. subpiraticus and P. pseudoannulata were the dominant species at the experimental site [70], but the number of trapped spiders was lower than expected. There are many uncontrollable factors that affect spider numbers, such as temperature, humidity, wind, the size and location of the trap, and the concentration of attractants.
In conclusion, our results indicate that HIRVs can attract spiders and increase predation. Two dominant species of rice spiders (P. pseudoannulata and P. subpiraticus) were attracted by one or two concentrations of methyl salicylate, linalool, or 2-heptanone. A predatory ability experiment showed that linalool and methyl salicylate shortened predation latency. Linalool increased the daily predation capacity of the spiders. A field experiment verified that methyl salicylate and linalool are attractive to spiders. In future research, we will determine the reasons why HIRVs enhance the predation ability of spiders and develop practical methods for using HIRVs to attract and retain spiders in rice fields.