Use of Food Attractant to Monitor and Forecast Population Dynamics of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), a Long-Distance Migratory Pest

: The rice leaf folder, Cnaphalocrocis medinalis , is a signiﬁcant migratory pest that restricts rice production in Asia and Africa, and monitoring and early warning measures are the basis for its management. Based on its regional migratory path in China, the pest population dynamics were monitored from 2020 to 2021 using food attractants. In this study, we also used internal reproductive system dissection to determine the adult age and reproductive developmental state. The monitoring results indicated that the trapping dynamics of C. medinalis males using food attractants and sex pheromones were approximately similar. Both trapping methods yielded C. medinalis males of different ages, with an identical age structure for both trap types. Dissection analysis of the reproductive system of females trapped using the food attractants indicated that in 2020, the population of C. medinalis in Liling, Hunan Province, was indigenous, while populations in other monitoring sites mainly comprised migrants from other areas. The ovarian development level of females trapped using food attractants showed a positive correlation with the age of males, and there was also a signiﬁcant positive correlation between the index of ovarian development of females and the mating time. Furthermore, a dynamic prediction method for fecundity in ﬁeld populations was established based on the quantity of eggs held by the females. This study provides a new method for the monitoring and early warning of the existence of C. medinalis populations.


Introduction
Rice is one of the world's major food crops, with about 3 billion people worldwide relying on rice as a staple food. The rice leaf folder, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), is one of the most significant pests affecting rice. The larvae prefer to feed on young rice leaves, often causing white streaks on the surface, which affects leaf photosynthesis, leading to an increase in the rate of rice blight and a decrease in grain weight [1][2][3]. Frequent outbreaks of C. medinalis in many Asian countries such as China, India, and Japan have caused huge losses to Asian rice production [4][5][6][7][8]. C. medinalis caused 5%-25% annual rice losses in Bangladesh [9]. From 1991 to 2019, the average annual area of occurrence of C. medinalis in China was 16.05 × 10 8 ha, accounting for 66.3% of the rice planting area, resulting in a rice loss of 70.27 × 10 8 kg [10,11].
C. medinalis has a long-distance migratory habit; Fu et al. used radar to monitor the migration dynamics of C. medinalis in the Bohai Bay area [12] and provided the flight speed, The C. medinalis food attractant (ingredients and ratio: methyl salicylate:limonene: camphor = 1:0.5:2.5) was a solid block [28], with an active ingredient content of 4 g/package. C. medinalis sex pheromones were composed of Z13-18: Ald, Z11-18: Ald, Z13-18: OH, and Z11-18: OH, at a 500:60:120:60 ratio [14], with 740 µg/pc active ingredient provided on a PVC capillary. Bell inverted funnel traps were fixed at 50 cm above the rice plant, and the height was adjusted regularly with the growth of the plant. The food attractants were replaced once every 15 days, and the sex pheromones were changed once every 30 days. The bell inverted funnel traps, food attractants, and pheromones were provided by Shenzhen Bioglobal Agricultural Science Co., Ltd. (Shenzhen, China). This is a company focused on green pest management, and its food attractant products for use with agricultural pests, such as Helicoverpa armigera and Spodoptera frugiperda, indicate good prospects for pest monitoring [30,31].
The number of adult C. medinalis females and males per trap was investigated every morning, and all trapped C. medinalis were transferred to the laboratory for dissection and determination of reproductive development status. On a daily basis, up to 50 male and female individuals (per type of attractant) were dissected, and all were dissected if the trapping number was less than 50.

Methods of Adult Dissection and Determination of the Age of Males
Referring to the anatomical methods of Feng et al. [23], adult C. medinalis trapped with food attractants and sex pheromones in the field were dissected every day. The adult abdomen was cut with a pair of scissors (12 cm long), placed in a Petri dish (5 cm diameter), and the abdominal body wall was torn through the fracture under a TS75-X trinocular microscope (Shanghai Shangguang New Optical Technology Co., Ltd. Shanghai, China) to separate the ovaries ( Figure 1A) and testes ( Figure 1D). Next, the testes were removed and placed in a 5 cm diameter plastic Petri dish filled with saline solution. The Petri dish was placed under a stereoscope (TS-75X; Shanghai Shangguang New Optical Technology Co., Ltd., Shanghai, China), and the major axis length of the testis (i.e., testis size) was measured with an OLD-SGD imaging system (Shanghai Shangguang New Optical Technology Co., Ltd.). The age of the males was judged by referring to the dividing criteria of C. medinalis (Table 2) suggested by Chen et al. [32]. After completion of the ovarian dissection, the index of ovarian development and the number of mating times (number of spermatophore in the bursa copulatrix) were determined and recorded by referring to the ovarian development classification criteria of Zhang et al. [33]. The number of spermatophores ( Figure 1C) in the bursa copulatrix ( Figure 1B) of female C. medinalis can indicate the number of mating times. That is, the presence of X spermatophores in the bursa copulatrix indicates mating X times [23]. tion, the index of ovarian development and the number of mating times (number of spermatophore in the bursa copulatrix) were determined and recorded by referring to the ovarian development classification criteria of Zhang et al. [33]. The number of spermatophores ( Figure 1C) in the bursa copulatrix ( Figure 1B) of female C. medinalis can indicate the number of mating times. That is, the presence of X spermatophores in the bursa copulatrix indicates mating X times [23].

Determination of Adult Source Properties and Dynamic Prediction of Oviposition
Referring to Zhang et al. [33], the property of insect origin of C. medinalis was determined by the following criteria: if the proportion of individuals with an index of ovarian development of 1 regarding females trapped using food attractants is 10%-30%, and the proportion of individuals with an index of ovarian development of 3-5 is 30%-62.18% during the monitoring period, then the population of C. medinalis at the experimental site

Determination of Adult Source Properties and Dynamic Prediction of Oviposition
Referring to Zhang et al. [33], the property of insect origin of C. medinalis was determined by the following criteria: if the proportion of individuals with an index of ovarian development of 1 regarding females trapped using food attractants is 10%-30%, and the proportion of individuals with an index of ovarian development of 3-5 is 30%-62.18% during the monitoring period, then the population of C. medinalis at the experimental site is a native population. If the proportion of individuals with an index of ovarian development of 3-5 of females trapped using food attractants is about 80% or more, the population of C. medinalis at the experimental site is mainly a migrant population from other regions.
Prediction of egg-laying dynamics of females: based on the ovarian development law of females of C. medinalis [33], the females exhibit the highest amount of egg holding when the ovaries develop to grade 3, and this result can be used as the total egg amount of the females before egg laying. The egg yield of a female in the field on a given day can be calculated using the following formula: p = 1 − X N . In the formula, p is the percent of oviposition of females in the field on a given day; X is the average number of eggs held by females trapped using food attractants on a certain day; N is the average egg holding capacity of C. medinalis trapped in the field with an index of ovarian development of 3. The daily percent of oviposition of females during the monitoring period was calculated to obtain the oviposition dynamics of females in the field.

Statistical Analysis
A Student's t-test was used to compare the daily trap counts between traps with food attractants and those with sex pheromones. A Chi-square test was used to assess differences in the sex ratio of female to male (1:1), as well as the male age structure for individuals caught in traps using food lures or sex pheromones. Pearson correlation analysis was used to compare the index of ovarian development, age of males, and number of matings. All data were analyzed using SPSS 26.0 (IBM, Armonk, NY, USA).

Comparison of Monitoring Effects of Food Attractants and Sex Pheromones
The peak in both the food attractant and sex pheromone mediated monitoring of C. medinalis in Liling occurred in mid-July (Figure 2A), while the peak of both food attractant and sex pheromone monitoring of C. medinalis in Zhangjiagang occurred twice: once in late July and once in late August ( Figure 2B). At the two sites, the peak occurrence of C. medinalis was essentially the same using either of the two trapping methods. At Shaodong in 2021, C. medinalis monitored using food attractants showed one peak period in early July, and those monitored using sex pheromones also exhibited one peak period in early July ( Figure 2C). The peak period of C. medinalis was essentially the same for both food attractant and sex pheromone monitoring, but the peak period for sex pheromone monitoring was longer, and the number of trappings was higher in early and mid-July.
In 2020 in Liling, a total of 4142 adult C. medinalis were trapped using food attractants, and 253 adult C. medinalis were trapped using sex pheromones. A total of 2569 adults were trapped using food attractants, and 211 adults were trapped using sex pheromones in Zhangjiagang. Food-based trapping thus yielded a higher daily trap capture than pheromone-based approaches in Liling and Zhangjiagang (Liling: t = 8.46, df = 60, p < 0.05; Zhangjiagang: t = 6.81, df = 48, p < 0.05). In 2021, a total of 6615 adults were trapped using food attractants, and 6429 adults were trapped using sex pheromones. There was no significant difference in the average daily trapping amount of C. medinalis using food attractants or sex pheromones in Shaodong. (t = −1.93, df = 86, p = 0.06).
The anatomical results regarding C. medinalis males trapped during the year 2020 using food attractants and sex pheromones showed that the highest percentage of 1-day-old males trapped using food attractants and sex pheromones in Liling was 17.8% and 23.72%, respectively. The lowest percentage of 7-day-old males trapped using food attractants was 8.67%, and the lowest percentage of 8-day-old moths trapped using sex pheromones was 5.53% ( Figure 2D). The highest proportion of 8-day-old males trapped using food attractants and sex pheromones in Zhangjiagang was 35.71% and 32.65%, respectively. The lowest proportion of 1-day-old males trapped by food attractants and sex pheromones was 1.02% and 2.04%, respectively ( Figure 2E). Both food attractants and sex pheromones trapped males at almost all ages (i.e., 1-8 days old), and there was no significant difference in the age structure of males trapped using either trapping method (Liling: χ 2 = 13.33, df = 7, p = 0.064; Zhangjiagang: χ 2 = 9.264, df = 7, p = 0.234). The highest percentage of 8-day-old males trapped using food attractants and sex pheromones in Shaodong in 2021 was 27.50% and 22.50%, respectively, while that of 1-day-old males was the lowest, with both capturing 4% ( Figure 2F). The age structure of field-caught males did not differ between either trapping method (Shaodong: χ 2 = 4.482, df = 7, p = 0.722).
In 2020, the results of the field monitoring using food attractants in seven different locations showed significant differences in the number of females and males of C. medinalis captured in different locations. With the exception of Zhangjiagang, the number of females was higher than the number of males, and the number of males in the remaining six sites was significantly higher than the number of females (Table 3). In 2021, the number of males Agronomy 2023, 13, 2141 6 of 16 trapped by C. medinalis was significantly higher than that of females at the two sites, except for the number of females and males trapped using food traps in Taihe, which was not significantly different (Table 3).
Agronomy 2023, 13, x FOR PEER REVIEW 6 of 16 difference in the age structure of males trapped using either trapping method (Liling:  2 = 13.33, df = 7, p = 0.064; Zhangjiagang:  2 = 9.264, df = 7, p = 0.234). The highest percentage of 8-day-old males trapped using food attractants and sex pheromones in Shaodong in 2021 was 27.50% and 22.50%, respectively, while that of 1-day-old males was the lowest, with both capturing 4% ( Figure 2F). The age structure of field-caught males did not differ between either trapping method (Shaodong:  2 = 4.482, df = 7, p = 0.722). In 2020, the results of the field monitoring using food attractants in seven different locations showed significant differences in the number of females and males of C. medinalis captured in different locations. With the exception of Zhangjiagang, the number of females was higher than the number of males, and the number of males in the remaining six sites was significantly higher than the number of females (Table 3). In 2021, the number of males trapped by C. medinalis was significantly higher than that of females at the two sites, except for the number of females and males trapped using food traps in Taihe, which was not significantly different (Table 3).

Monitoring Seasonal Population Migration Dynamics of C. medinalis Using Food Attractants
During the monitoring period in 2020, the C. medinalis peak occurred in mid to late July ( Figure 3B-D), except for Guigang, in which the peak of C. medinalis was observed in early June ( Figure 3A). The maximum number of trapped C. medinalis was 1631/day in Shaodong, and the lowest was 220/day in Quzhou.

Monitoring Seasonal Population Migration Dynamics of C. medinalis using Food Attractants
During the monitoring period in 2020, the C. medinalis peak occurred in mid to late July ( Figure 3B-D), except for Guigang, in which the peak of C. medinalis was observed in early June ( Figure 3A). The maximum number of trapped C. medinalis was 1631/day in Shaodong, and the lowest was 220/day in Quzhou. In 2021, the peak periods of C. medinalis at Shaodong and Zhijiang were basically the same ( Figure 3E). Shaodong recorded the highest number of C. medinalis trapped on June 30, with a maximum of 883 adults per day. Zhijiang reached its highest trapping number of C. medinalis on 2 July, with a maximum of 136/day. At Taihe, there was a peak of C. medinalis in early June, and the highest trapping number was reached on June 5, with a maximum of 117/day ( Figure 3E).

Monitoring the Reproductive Development Status of C. medinalis using Food Attractants
The anatomical results of females trapped using food attractants in different locations in 2020 showed that the proportion of individuals with an index of ovarian development of 1 in females trapped using food attractants in Guigang was 11.96%, and that of individuals with an index of ovarian development of 3-5 was 82.68% ( Figure 4A). The proportion of individuals with an index of ovarian development of 1 trapped using food attractants in Liling was 10.92%, and the proportion of individuals with an index of ovarian development of 3-5 was 62.78% ( Figure 4B). The percentage of individuals with an index of ovarian development of 1 and 3-5 in Shaodong was 0.77% and 94.59%, respectively ( Figure 4C). In Changzhou, the proportion of trapped females with an index of ovarian development of 1 and 3-5 was 0 and 94.59%, respectively ( Figure 4D). In Zhangjiagang, the proportion of trapped females with an index of ovarian development of 1 and 3-5 was 2.88% and 94.01%, respectively ( Figure 4E). In Quzhou, the proportion of trapped females with an index of ovarian development of 1 and 3-5 was 9.30% and 77.41%, respectively ( Figure 4F). In Xiangshan, the proportion of trapped females with an index of ovarian development of 1 and 3-5 was 1.60% and 94.57%, respectively ( Figure 4G). Based on the criteria for determining the nature of C. medinalis origin, it can be judged that in 2020, except for the Liling C. medinalis population as a local population, the populations of C. medinalis in the other six locations were mainly migrant species.

Prediction of Oviposition Dynamics of C. medinalis Based on Food Attractants Monitoring Data
In 2020, the egg holding amount of C. medinalis in Guigang was low during the monitoring period. The lowest egg holding volume was 9.80 ± 5.07 on May 29, and the highest egg holding volume was 56.00 ± 4.00 on June 21 ( Figure 7A). The prediction of oviposition dynamics showed that the highest percentage of oviposition in Guigang was 86.00% on May 29, and the lowest was 20.00% on 21 June (Figure 7A). At Liling in 2020, the lowest egg holding capacity was 21.67 ± 3.09 on 9 July, and the highest egg holding capacity was 40.35 ± 2.65 on July 19 ( Figure 7B). Liling showed a maximum percentage of oviposition of 69.05% on 9 July and a minimum percent oviposition of 42.35% on 19 July ( Figure 7B).

Prediction of Oviposition Dynamics of C. medinalis Based on Food Attractants Monitoring Data
In 2020, the egg holding amount of C. medinalis in Guigang was low during the monitoring period. The lowest egg holding volume was 9.80 ± 5.07 on May 29, and the highest egg holding volume was 56.00 ± 4.00 on June 21 ( Figure 7A). The prediction of oviposition dynamics showed that the highest percentage of oviposition in Guigang was 86.00% on May 29, and the lowest was 20.00% on 21 June (Figure 7A). At Liling in 2020, the lowest egg holding capacity was 21.67 ± 3.09 on 9 July, and the highest egg holding capacity was 40.35 ± 2.65 on July 19 ( Figure 7B). Liling showed a maximum percentage of oviposition of 69.05% on 9 July and a minimum percent oviposition of 42.35% on 19 July ( Figure 7B).

Discussion
The real-time and accurate monitoring of the population dynamics of migratory pests can provide important data for prediction and control decisions, laying a solid foundation for integrated pest management concepts [34,35]. This study showed that the monitoring

Discussion
The real-time and accurate monitoring of the population dynamics of migratory pests can provide important data for prediction and control decisions, laying a solid foundation for integrated pest management concepts [34,35]. This study showed that the monitoring of insect age and reproductive development state is possible using food attractant traps. We also showed how reproductive system analysis can be used to determine the nature of the origin of C. medinalis at different sites. In addition, we investigated the relationship between ovarian development, male age, and mating time of adult C. medinalis trapped using food attractants and predicted the fecundity dynamics of C. medinalis. In addition, in this study, a new monitoring and early warning method based on food attractants trapping of C. medinalis was explored, which is valuable for guiding the regional management of this pest.
In this study, the monitoring effects of the use of food attractants and sex pheromones on C. medinalis were compared, and the results showed that the peak occurrence period of adults trapped using food attractants and sex pheromones in three different locations was basically consistent during the two-year monitoring period. However, the trapping efficiency of food attractants was superior to that of sex pheromones. For example, in 2020, the number of adults trapped using food attractants in Liling and Zhangjiagang was significantly higher than that obtained using sex pheromones. Zeng et al. trapped adult C. medinalis in different ways, and found that the number of adults trapped using food attractants was higher than that obtained using sex pheromones, and this method could trap both sexes simultaneously [28]. This result is consistent with the results of the current study using food attractants to monitor C. medinalis. We also compared the age structure of males trapped using food attractants and sex pheromones and found that almost all males of each age (1-8 days old) could be trapped using either trapping method, and there was no significant difference in the age structure of males between the two methods. This is consistent with the results obtained by He et al. using food attractants to trap adult S. frugiperda [31]. Different food attractants often result in different sex ratios when trapping different species of pests. For example, the sex ratio of Spodoptera exigua trapped using food attractants was close to 1:1 [36], while the number of female S. frugiperda trapped was significantly higher than the number of male moths [31]. This may be caused by the different volatile components of food attractants. Cheng et al. found that male C. medinalis showed strong potential responses to n-valeraldehyde, benzaldehyde, and methyl salicylate [26]. In addition, based on the monitoring results of food attractants in 2020-2021, it can be seen that the peak occurrence period of C. medinalis in different locations in the same province was basically the same. For example, in 2020, the peak periods of C. medinalis in Liling and Shaodong in Hunan Province, Changzhou and Zhangjiagang in Jiangsu Province, and Quzhou and Xiangshan in Zhejiang Province are basically the same, and the peak periods of C. medinalis in Shaodong and Zhijiang in Hunan Province in 2021 was basically the same. We infer that C. medinalis may migrate to different locations in the same province on a large scale at similar times. Therefore, the control strategy for C. medinalis must not only be limited to a single area, but needs to be managed in the pest source province for reducing its possibility of regional outbreaks by long distance migration. Insect ovary dissection technology is one of the most important methods for studying pest occurrence patterns and predicting their times of onset. By the dissection of female ovaries in the field, the process of ovarian development of females can be determined, and subsequently, the nature of insect origin can be determined [37]. In this study, by dissecting the ovaries of females trapped using food attractants, it was found that the sources of trapped C. medinalis were different in different years and places. For example, in 2020, the population of C. medinalis in Liling was a native population, and the populations of C. medinalis in the rest of the sites were mainly migrant populations from other regions. According to the migration principle, C. medinalis began migrating north from Guangxi and Hainan Province from late May to mid-June, mainly landing in the Jiangling rice area in eastern and northern China. In mid-to-late June, a large number of C. medinalis migrated from the Jiangling rice area to the vast Jianghuai rice area in the Huaihe River Basin and the Hanzhong Basin. From late July to early August, it continued to move north, reaching the northern rice areas of North and Northeast China. From late August to early September, C. medinalis began to migrate southward to the perennial rice areas of Jianghuai, Jiangling, and Hainan [38,39]. In this study, adult C. medinalis monitored in Hunan, Guangxi, Jiangsu, Zhejiang, and Jiangxi provinces were predominantly migrant populations, which were consistent with the C. medinalis north-south migration law in China. This study also showed that ovarian development was positively correlated with both the moth age and mating times of C. medinalis. Therefore, when the number of females trapped using food attractants is low, the ovarian development level of females can be inferred based on the age of male C. medinalis to accurately monitor the reproductive development status of adult C. medinalis. When anatomical samples are damaged, the accuracy of determining the ovarian development index can be improved by analyzing the mating time of female C. medinalis. Based on the egg holding capacity of female C. medinalis trapped in the field, we can also speculate on the percentage of oviposition of the C. medinalis population in the field, which is helpful in establishing a larval hatching model.
In this study, by analyzing the reproductive development status of adult C. medinalis trapped in the field using an attractant and number dynamics, the origin of the field population of C. medinalis can be more accurately explored, and fecundity dynamics can be predicted. In the future, for comprehensive control of C. medinalis, the monitoring of food attractants may be combined with physical control, and light trapping may be used to trap adults during peak periods [40]. Based on the analysis of the nature of the insect source, trapping measures along the migration path may be established to reduce migration in crop growing areas. In addition, the combination of the use of food attractants and push-pull strategies may reduce the infestation of C. medinalis, i.e., in field trials, by combining food attractants and push-pull strategies to reduce the fecundity of Drosophila suzukii and Delia radicum [41][42][43].
Similar to the results of this study, the use food attractants has also showed a strong ability to monitor other pests. For example, the use of food attractants (pear esters) in apple orchards can effectively capture female and male codling moths, and the trapping peak for the use of food attractants is consistent with those obtained using sexual attractants [44,45]. However, trapping efficiency was often low regarding the use of food attractants. For example, the average daily fall armyworm collection number was only 0.5/trap [29], and the average daily trapping number of females and males of the grape berry moth was less than 1/trap [46]. In this study, the average daily number pests trapped using food attractants was higher than that obtained using sex pheromones, providing more accurate monitoring results. Moreover, since food attractants are usually composed of plant volatiles, they are also attractive to non-target insects. For example, food attractants often trap beneficial insects, while monitoring population dynamics [47]. In this study, the proportion of C. medinalis trapped using food attractants in the field monitoring process was as high as 90%, and this method was also highly specific and less harmful to non-target insects, such as beneficial insects.
Food attractants are convenient and environment friendly, but they still exhibit some problems. Under field conditions, the effective period for C. medinalis food attractants are short, most of which are only 15-20 d [48]. Moreover, the high concentration of volatiles in food attractants at the early stage of the test resulted in some repellent effect on C. medinalis, while the decreased volatile concentration at the later stage of use reduced adult trapping, which had a greater impact on the long-term monitoring of population dynamics. Factors such as the color, shape, and height of the trap may also have an impact on trapping efficiency. Therefore, in order to improve the monitoring accuracy of food attractants, further studies are needed in terms of food attractant carrier materials, volatile compound field release rate, trap settings, and field distribution. This study showed that food attractants can accurately monitor C. medinalis population dynamics in the field. Combined with reproductive anatomy techniques, the reproductive development status and origin characteristics of C. medinalis in field populations can be further analyzed. In addition, we have established a link between the reproductive development of females and males trapped using food attractants, which can improve the accuracy of monitoring. Our research is useful in advancing the development of field population surveillance technology for C. medinalis, which is of great value to guide the regional management of this pest.