Escuela Agrícola Panamericana, Zamorano Agricultural Science and Production B.S. in Agricultural Sciences Special Graduation Project Evaluation of Two Entomopathogenic Nematodes Heterorhabditis bacteriophora (H. K. Poinar) and Steinernema carpocapsae (J. Weiser) Using Two Applications Methods for the Control of Spodoptera frugiperda (J.E. Smith) in Maize Production Students Adrián José Hernández Sánchez Giorgio Brustolin Zelaya Flores Advisors Rogelio Trabanino, M.Sc. Miguel Cocom, B.Sc. Erick Figueroa, B.Sc. Honduras, November 2024 2 Authorities SERGIO ANDRÉS RODRÍGUEZ ROYO President ANA M. MAIER ACOSTA Vice President and Academic Dean CELIA O. TREJO RAMOS Director of Agricultural Science and Production Department JULIO NAVARRO Secretary General 3 Table of Content List of Tables ............................................................................................................................................ 4 List of Figures .......................................................................................................................................... 5 List of Appendices ................................................................................................................................... 6 Abstract ................................................................................................................................................... 7 Resumen ................................................................................................................................................. 8 Introduction ............................................................................................................................................ 9 Materials and Methods ......................................................................................................................... 11 Location ................................................................................................................................................. 11 Experimental Location .......................................................................................................................... 12 Pest Infestation ..................................................................................................................................... 12 Treatments and Applications ................................................................................................................ 12 Measured Variable ................................................................................................................................ 14 Experimental Design and Statistical Analysis ........................................................................................ 14 Results and Discussion .......................................................................................................................... 15 Data Analysis ......................................................................................................................................... 15 S. frugiperda’ s Mortality Rate Found 48 Hours After Treatment Application ...................................... 18 S. frugiperda’ s Mortality Rate Found 72 Hours After Treatment Application ...................................... 20 Conclusions ........................................................................................................................................... 22 Recommendations ................................................................................................................................ 23 Referencias ............................................................................................................................................ 24 Appendices ............................................................................................................................................ 26 4 List of Tables Table 1 Description of treatments for evaluating two entomopathogenic nematodes, H. bacteriophora and S. carpocapsae, and two application methods for the control of Spodoptera frugiperda in maize production. ............................................................................................................................................ 12 Table 2 Factorial arrangement results of the evaluation of two entomopathogenic nematodes, H. bacteriophora, and S. carpocapsae, and an Emamectin Benzoate insecticide with two application methods to control Spodoptera frugiperda in maize production. ........................................................ 15 Table 3 Means mortality percentages for S. frugiperda controllers from the evaluation of two entomopathogenic nematodes, H. bacteriophora, and S. carpocapsae, and an Emamectin Benzoate insecticide with two application methods to control Spodoptera frugiperda in maize production. .... 16 Table 4 Means mortality percentages for application methods from the application methods from the evaluation of two entomopathogenic nematodes, H. bacteriophora and S. carpocapsae, using two application methods for the control of Spodoptera frugiperda in maize production. ......................... 17 Table 5 Mean mortality percentages for sampling times from the evaluation of two entomopathogenic nematodes, H. bacteriophora, and S. carpocapsae, using two application methods for the control of Spodoptera frugiperda in maize production. ........................................................................................ 17 Table 6 Mortality rate on S. frugiperda at 48 and 72-hours post-treatment application from the evaluation of two entomopathogenic nematodes, H. bacteriophora and S. carpocapsae, using two application methods in maize production. ........................................................................................... 19 5 List of Figures Figure 1. Biological Control Research Unit in the Pan-American Agricultural School, Zamorano. ...... 11 Figure 2 Location of the evaluated treatments in the Biological Control Unit, Zamorano Honduras. . 13 6 List of Appendices Appendix A Application Tools ................................................................................................................ 26 Appendix B Backpack Sprayer Application Method .............................................................................. 27 Appendix C Drone Application Method ................................................................................................ 28 Appendix D Evaluation of Nematode Infection. .................................................................................... 29 Appendix E Nematodes on Fall Armyworm. ......................................................................................... 30 Appendix F Precipitation During the Experiment ................................................................................. 31 Appendix G Diet ingredients for S. frugiperda. ..................................................................................... 32 7 Abstract One of the most critical pests that significantly threatens maize crops is Spodoptera frugiperda, which can cause up to 50% production losses. This experiment aimed to compare the efficacy of the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae against the insecticide emamectin benzoate, applied using a DJI© Agras T10 spray drone and a conventional HIDRA PROTECNO 16 backpack sprayer for the control of S. frugiperda. The mortality of S. frugiperda larvae was evaluated 48 and 72 hours after application, and data were analyzed using SAS statistical analysis software version 9.4 4 through an analysis of variance (ANOVA) and Duncan´s new multiple range test for means comparisons. Results showed that at 72 hours post-application, there was a higher mortality of S. frugiperda across all treatments, with the chemical treatment applied by drone achieving the highest mortality at 90%, followed by 68% with the backpack sprayer. Nematode treatments with the backpack sprayer reached a mortality rate of 42% for S. carpocapsae and 36% for H. bacteriophora. In the drone applications, the nematode S. carpocapsae achieved 26% mortality, and H. bacteriophora had a 2% mortality rate. Keywords: backpack sprayer, drone, effectiveness, mortality. 8 Resumen Una de las plagas más críticas que amenaza significativamente el cultivo de maíz es Spodoptera frugiperda, que puede causar pérdidas de hasta el 50% de la producción. Este experimento tuvo como objetivo comparar la eficacia de los nematodos entomopatógenos, Heterorhabditis bacteriophora y Steinernema carpocapsae, contra el insecticida benzoato de emamectina, aplicados mediante un dron de fumigacion DJI© Agras T10 y un pulverizador de mochila convencional HIDRA PROTECNO 16 para el control de S. frugiperda. Se evaluó la mortalidad de larvas de S. frugiperda a las 48 y 72 horas después de las aplicaciones; estos datos se analizaron utilizando el sistema de análisis estadístico SAS software versión 9.4 mediante un analisis de varianza y la nueva prueba de rango múltiple de Duncan para comparaciones de medias. Se determinó que a las 72 horas post aplicación, hubo una mayor mortalidad de S. frugiperda, en todos los tratamientos, siendo el tratamiento químico aplicado con dron el de mayor mortalidad, con un 90% y el segundo con un 68% con el pulverizador de mochila. Los tratamientos contra nematodos con pulverizador de mochila alcanzaron una mortalidad del 42% para S. carpocapsae y del 36% para H. bacteriophora. En las aplicaciones con dron, el nematodo S. carpocapsae obtuvo un 26% , y H. bacteriophora tuvo un 2% de mortalidad. Palabras clave: dron, eficacia, mortalidad, pulverizador de mochila. 9 Introduction One of the most critical pests in maize production is the fall armyworm (Spodoptera frugiperda), a lepidopteran of the noctuid family; it can cause losses of up to 50% of production. This pest has a short life cycle that lasts approximately 24 to 40 days (Gobierno de Nueva Gales del Sur [NSW], 2024; Proain Tecnología Agrícola [PROAIN], 2020). Environmental temperature can affect S. frugiperda´s life span; temperatures of 28 to 30°C shorten their life cycle, and temperatures of 21 to 23 °C lengthen it. S. frugiperda, in the larvae stage, attacks maize from germination to crop maturity. Early attacks can affect vegetative development stages, while late attacks can damage spikes and cobs (Lezaun, 2022). Larvae feed on all aerial plant tissues, such as young leaves and whorls, affecting photosynthesis and causing structural damage, and on reproductive tissues, such as maize ears and tassels, directly impacting yields (Chimweta et al., 2019). The most common method of controlling pests is using chemical insecticides. However, the overuse of selective insecticides will increase resistance levels in S. frugiperda (NSW, 2024). Indiscriminate use of pesticides has the disadvantage of killing non-target insects, including natural enemies of the intended pest (Matova et al., 2020). Sustainable and environmentally friendly agricultural practices are becoming popular today, allowing farmers new strategies to control them. A less explored but promising biocontrol strategy is using entomopathogenic nematodes (EPNs), such as Heterorhabditis bacteriophora and Steinernema carpocapsae, to manage lepidopteran pests (Ballal et al., 2021). H. bacteriophora is an obligate parasitic nematode mutually associated with the enteric bacterium Photorhabdus luminescens. This bacterium is stored in the nematode's intestine and remains dormant until an infective juvenile (IJ) nematode enters a host. Its life cycle begins as an infective juvenile (IJ) that actively seeks a host, which it enters through natural openings, such as the anus, mouth, or spiracles (Todd Ciche, 2007). After finding and penetrating the host, the nematode 10 regurgitates P. luminicens, which begins to reproduce rapidly and releases toxins and enzymes that help degrade the host's tissues, turning the host into a nutrient medium for both the P. luminicens bacteria and the H. bacteriophora nematode, which begins to feed and reproduce inside the host's corpse and produce more IJs, which will continue with a new life cycle (Ciche y Ensing, 2003). S. carpocapsae has a similar method of infection, with the IJ being responsible for searching and penetrating the host, and is associated with the bacterium Xenorhabdus nematophila, which performs the same function as P. luminicens with H. bacteriophora (Lefoulon et al., 2022) . Although these organisms have similar modes of action, they differ in infection strategies; S. carpocapsae waits for a host to pass near it, while H. bacteriophora is more active in searching for hosts (Serra et al., 2019). The virulence of H. bacteriophora and S. carpocapsae decreased in field trials where these nematodes encountered many adverse factors affecting their survival and efficacy in controlling pests. Overall, this study provides an alternative entomopathogenic nematode (EPN) for the biological control of S. frugiperda, wich has the potential to be developed as a sustainable option for efficient pest management (Chen et al., 2022). Applying beneficial nematodes is straightforward and ensures contact with target insects. Nematodes can be applied to foliage or soil depending on the target pest (Koppert [K], 2024). Standard nematode application tools include manual backpack sprayers, drones, spraying robots, tractor spray tanks, irrigation systems, and atomizer blowers. This study aims to determine the effectiveness of nematodes H. bacteriophora and S. carpocapsae in controlling Spodoptera frugiperda larvae using a drone and a backpack sprayer as application methods. 11 Materials and Methods Location The experiment was carried out in the Biological Control Unit of the Zamorano Panamerican Agricultural School (Figure 1), which is located 30 km from Tegucigalpa, Francisco Morazán department. The site is 14° north latitude, 87° west longitude, 800 meters above sea level. During the experiment, the precipitation was 344.6 mm, and the average temperature was 23 °C. The experiment began on May 24 and ended on July 24, 2024. Figure 1 Biological Control Research Unit in the Pan-American Agricultural School, Zamorano. Note. The division of every treatment is shown in Figure 2 12 Experimental Location The evaluation was conducted at the Biological Control Laboratory's experimental plot from May 24 to June 24. The plot was 2100 m2 and included 56 rows of maize 50 m long. The area was divided into 35 experimental units, each with eight rows of maize measuring 10 m long and 6 m wide ( Figure 2). Pest Infestation On June 21st, one day before the treatment application, each experimental unit was inoculated in the fourth and fifth rows of maize with third instar larvae of ten S. frugiperda in each row. The aim was to achieve a homogeneous infestation in each experimental unit, avoiding factors such as variability in the age of S. frugiperda larvae. The Zamorano´s Biological Control Unit laboratory provided the third instar larvae of S. frugiperda. Treatments and Applications Treatments consisted of two entomopathogenic nematodes (Nep´s), Heterorhabditis bacteriophora and Steinernema carpocapsae, and the chemical insecticide Denim Fit 45EC of active ingredients 5% Emamectin Benzoate and 40% Lufenuron to control S. frugiperda. Treatments were applied using a DJI© Agras T10 drone and a Protecno Hidra 16 backpack sprayer. The concentration of entomopathogenic nematodes and the chemical treatments are shown in Table 1. Table 1 Description of treatments for evaluating two entomopathogenic nematodes, H. bacteriophora and S. carpocapsae, and two application methods for the control of Spodoptera frugiperda in maize production. Treatments Method Dosage¥ Heterorhabditis bacteriophora Drone 200 million/ha Steinernema carpocapsae Drone 200 million/ha Emamectin Benzoate and Lufenuron Drone 1 g/ L 13 Treatments Method Dosage¥ Heterorhabditis bacteriophora Backpack Sprayer 200 million/ha Steinernema carpocapsae Backpack Sprayer 200 million/ha Emamectin Benzoate and Lufenuron Backpack Sprayer 1 g/L Untreated Backpack Sprayer water Note. ¥Concentration of entomopathogenic nematodes used was selected from past evaluations in the Biological Control Unity. The Emamectin Benzoate and Lufenuron treatment concentration was extracted from the product's technical sheet. Treatment applications were done at 6 a.m. to prevent wind drift. Applications made using a DJI© Agras T10 drone were performed using a flat fan nozzle in a volume application of 50 L/ha. The treatments applied using a backpack sprayer PROTECNO employed a hollow cone nozzle and a volume application of 300 L/ha. Figure 2 Location of the evaluated treatments in the Biological Control Unit, Zamorano Honduras. Note. The picture was taken with an unmanned aerial vehicle (UAV) with a multispectral camera. 14 Measured Variable Mortality rate of S. frugiperda larvae in maize plants was evaluated. The mortality was determined by collecting ten larvae per experimental unit 48 hours post-treatment application and transferring them in plastic containers to the Zamorano Biological Control laboratory with a special diet made of rice, beans, vitamins, and other ingredients (Appendix 7). The assessment of mortality was conducted in the 48 hours post-treatment application. Dead larvaes treated with nematodes were dissected and examined under a stereoscope to determine the presence of nematodes. Live larvae were kept in the laboratory for a subsequent assessment 72 hours post-treatment application. Experimental Design and Statistical Analysis The experiment used a Randomized Complete Block Design (RCBD) with a factorial arrangement of 3 × 2 × 2, with three treatments ( H. bacteriophora, S. carpocapsae, and Emamectin), two application methods (drone and backpack sprayer), and two sampling times (48 and 72-hours post-treatment application. Data were analyzed using SAS software version 9.4 through an analysis of variance (ANOVA) and Duncan´s new multiple range test for means comparisons. Mean separation was conducted using the least significant difference (LSD) test at a significance level of P ≤ 0.05. 15 Results and Discussion Data Analysis The results showed statistical differences between treatments, application methods, and sampling times; the analysis also showed interactions between the treatments and application methods (Table 2). No interaction was found between Treatments × Sampling Times, Application Methods × Sampling Times, and Treatments × Application Methods × Sampling Times (Table 2). Table 2 Factorial arrangement results of the evaluation of two entomopathogenic nematodes, H. bacteriophora, and S. carpocapsae, and an Emamectin Benzoate insecticide with two application methods to control Spodoptera frugiperda in maize production. The evaluated treatments presented significant differences. Emamectin and Lufenuron treatments reached the highest mortality value of 78.5%, Steinernema carpocapsae treatments got a mortality value of 38.5%, and Heterorhabditis bacteriophora treatment presented a mortality of 24.5% (Table 3). Source Mean Square F Value Pr > F Treatment (TRT) 1.57066667 104.71 <.0001 Applic. Method 0.1215 8.1 0.0065 TRT x Applic. Method 0.344 22.93 <.0001 Sampling time 0.08816667 5.88 0.0191 TRT x Sampling time 0.00866667 0.58 0.565 Applic. Method x Sampling time 0.00416667 0.28 0.6006 TRT x Applic. Method x Sampling time 0.00066667 0.04 0.9566 16 Table 3 Means mortality percentages for S. frugiperda controllers from the evaluation of two entomopathogenic nematodes, H. bacteriophora, and S. carpocapsae, and an Emamectin Benzoate insecticide with two application methods to control Spodoptera frugiperda in maize production. Results Treatments H. bacteriophora S. carpocapsae Emamectin and Lufenuron Mean mortality % 24% 38% 78% Standard Deviation 0.21 0.14 0.13 P Value <0.001 The higher mortality rate in the Emamectin and Lufenuron treatment may be due to their mode of action, which is by contact and translaminar effect, allowing more pathways to intoxicate the S. frugiperda larvae compared to entomopathogenic nematodes. S. carpocapsae showed a higher mortality rate in S. frugiperda larvae than H. bacteriophora, regardless of the application method. This aligns with findings by (Choo et al., 1991), who demonstrated in their experiment that S. carpocapsae performed better than H. bacteriophora in controlling forest pests in larval instars 1, 2, and 3. The application methods also presented significant differences; the data showed that the backpack sprayer application method achieved a mortality value of 51.6%, and the drone achieved a mortality value of 42.2% (Table 4). These data may be related to the higher focus achieved with applications made using the backpack sprayer, which were applied directly to the plant's whorl. In contrast, drone applications were less focused but covered a larger area. This affects the nematodes, as according to (Shapiro-Ilan et al., 2006), foliar applications of nematodes are less effective than soil applications. This is due to the lack of coverage against exposure to UV rays and the desiccation nematodes suffer from overexposure to solar radiation. 17 Additionally, (Catruita Esparza) mentions that including of adjuvants and protectants containing compounds that shield entomopathogenic nematodes from UV exposure and solar radiation can extend their lifespan up to 7 days, providing them with more time to infect pests. The two sampling times showed significant differences, having in the mortality percentage sampling 48 hours post-treatment application a mortality value of 43%, and on the 72 hours post- treatment application with a 51% mortality value (Table 5). Table 4 Means mortality percentages for application methods from the application methods from the evaluation of two entomopathogenic nematodes, H. bacteriophora and S. carpocapsae, using two application methods for the control of Spodoptera frugiperda in maize production. Table 5 Mean mortality percentages for sampling times from the evaluation of two entomopathogenic nematodes, H. bacteriophora, and S. carpocapsae, using two application methods for the control of Spodoptera frugiperda in maize production. Results Sampling Times 72 Hours 48 Hours Mean mortality % 51% 43% Standard Deviation 0.27 0.29 P Value 0.0065 Results Application methods Backpack sprayer Drone Mean mortality % 51% 42% Standard Deviation 0.18 0.35 P Value 0.0065 18 This difference in sampling times may be related to the fact that entomopathogenic nematodes need approximately 1.5 to 4 days to reproduce and kill their host (Molina Bustamante, 2022). S. frugiperda’ s Mortality Rate Found 48 Hours After Treatment Application The S. frugiperda larvae mortality evaluation 48 hours post-treatment application showed that 5% Emamectin and 40% Lufenuron applied using a drone presented a mortality rate of 86% (Table 6). According to (Somnath y Kamalkant, 2024) drone applications are less targeted but can cover more area, which is favorable for translaminar pesticides such as Emamectin and Lufenuron because the greater the foliar area covered during the application, the higher the percentage of protection against pests, as the insecticide penetrates the leaf and remains stored for a period of time, potentially intoxicating pests that feed on a leaf containing this pesticide. The chemical treatment applied with a backpack sprayer had a mortality rate of 68% (Table 6), indicating a decrease in effectiveness but outperforming nematodes treatments. It is essential to mention that no drift affected the application treatment effectiveness, and no adjuvant was used in the treatments; this can reduce the effectiveness of the nematode treatment applications. Among the biological treatments, S. carpocapsae, when applied using a backpack sprayer, reached a mortality rate of 42% (Table 6). In contrast, H. bacteriophora applied using a backpack sprayer had a slightly lower mortality rate of 36% (Table 6). However, the S. carpocapsae application using a drone showed reduced mortality percentage, with a 26% (Table 6). There were no significant differences between the S. carpocapsae x bp sprayer (ScBp) with H. bacteriophora x backpack sprayer (HbBp) and H. bacteriophora x backpack sprayer (HbBp) with S. carpocapsae x Drone (ScDn) treatments. The H. bacteriophora applied using a drone treatment showed the lowest effectiveness, with a mortality rate of only 2% (Table 6). There were no significant 19 differences between the H. bacteriophora using a drone (HbDn) and the control treatment, which had a mortality rate of 6%. The difference between the treatments (ScDn) and (HbDn) could be related to two factors. One is their different action mode according to (Tofangsazi et al., 2012) H. bacteriophora had a highly active searching behavior, moving a significant distance and looking for a host, while S. carporcapsae is an ambusher with an energy-conserving approach that lies in waiting to attack mobile insects, leaving the H. bacteriophora nematodes more susceptible to UV radiation and solar desiccation. Also (Tyson et al., 2007) found that S. carpocapsae has genes for resistance to desiccation and osmotic stress that are similar to those present in plants, which are not found in H. bacteriophora. This could be another possible reason for the lower mortality rate of S. frugiperda by H. bacteriophora. Table 6 Mortality rate on S. frugiperda at 48 and 72-hours post-treatment application from the evaluation of two entomopathogenic nematodes, H. bacteriophora and S. carpocapsae, using two application methods in maize production. Treatments Interaction Mortality at Mortality at 48 hours 72 hours (%) (%) Heterorhabditis bacteriophora Drone 2 e¥ 14 d Steinernema carpocapsae Backpack 42 c 48 c Heterorhabditis bacteriophora Backpack 36 cd 46 c Steinernema carpocapsae Drone 26 d 38 c 5% Emamectin and 40% Lufenuron x Backpack 68 b 70 b 5% Emamectin and 40% Lufenuron x Drone 86 a 90 a Untreated 6 e 12 d P Value < 0.0001 <0.0001 R2 0.91 0.86 CV 28.79 27.63 ¥Note. Means with different letters in each treatment are statistically different (P ≤ 0.05) with the Duncan test. 20 S. frugiperda’ s Mortality Rate Found 72 Hours After Treatment Application The 5% Emamectin and 40% Lufenuron treatment applied by drone showed a 4% increase in efficacy 72 hours after treatment application, achieving 90% mortality. Chemical treatment applied with a backpack sprayer indicated a 2% increase, resulting in a mortality of 70%. This chemical treatment demonstrated the highest mortality rate across both application methods, drone and backpack. However, there was a significant difference between treatments, with the drone application showing a 20% higher efficacy than the backpack sprayer application. Chemicals with translaminar and contact action, like emamectin benzoate, penetrate leaf tissue, making it effective for controlling lepidopteran larvae. This mode of action allows it to spread within plant tissues, targeting larvae that may be feeding beneath the surface. Such chemicals provide effective, long-lasting control by immobilizing pests shortly after ingestion or contact (Führ et al., 2024). The nematode treatments showed that both entomopathogenic species, H. bacteriophora and S. carpocapsae, when applied with the backpack method, were more effective in controlling S. frugiperda among the biological treatments. Specifically, S. carpocapsae achieved a mortality rate of 48%, while H. bacteriophora reached 46%. Additionally, the S. carpocapsae treatment via drone achieved a mortality rate of 38%. There were no significant differences among the three treatments in terms of S. frugiperda mortality. These results indicate that the S. carpocapsae entomopathogenic nematode treatment showed the highest mortality rate of S. frugiperda in both application methods. Similar results were presented by (Sayed et al., 2022) were the highest recorded mortality rate from 3, 4 and 5 larval S. frugiperda instars was 100% after 3 and 4 days of treatment application at a concentration of (80 IJs/ml) of irradiated S. carpocapsae nematodes, the recorded death rate for un- irradiated S. carpocapsae was 72.2 and 77.8% for the two treated larval instars, respectively, after 4 days of the treatment with the same concentration. 21 Heterorhabditis bacteriophora applied using a drone showed the lowest efficacy among all treatments, with a mortality rate of 14% , which was not significantly different from the untreated (Table 6). Mortality rate found in the control untreated is related to two natural enemies observed, a Hexamermis sp. parasite nematode and a parasitoid fly from the Tachinidae family naturally occurring. 22 Conclusions The S. carpocapsae nematode presented a better control of S. frugiperda than H. bacteriophora with both application methods. The 5% Emamectin and 40% Lufenuron treatment applied using a drone achieved the highest mortality percentage of S. frugiperda. The 72-hour sampling post-treatment application showed a higger mortality percentage than the 48-hour sampling post-treatment application. 23 Recommendations It is recommended that foliage application trials be conducted using adherent and protective agents that enhance the spread and survival of entomopathogenic nematodes after field application. Mitigating the damage caused by solar ultraviolet radiation and temperature is crucial, as these factors can negatively affect the viability and effectiveness of the nematodes. Diverse types of drone nozzles should be evaluated to ensure better coverage and penetration into the whorl,and preserve the viability of entomopathogenic nematodes. 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International Journal for Parasitology, 37(7), 763–776. https://doi.org/10.1016/j.ijpara.2006.12.015 26 Appendices Appendix A Application Tools 27 Appendix B Backpack Sprayer Application Method 28 Appendix C Drone Application Method 29 Appendix D Evaluation of Nematode Infection. 30 Appendix E Nematodes on Fall Armyworm. 31 Appendix F Precipitation During the Experiment Quarters (All) Sum of Rain Column Labels Row Labels 2024 January 0.2 February 0.6 March 24.2 April 20.4 May 10 June 334.6 July 186 August -- September -- October -- November -- December -- Grand Total 344.6 0 50 100 150 200 250 300 350 400 January February March April May June July Pr ec ip ita tio n (m m ) Year: 2024 32 Appendix G Diet ingredients for S. frugiperda. N Compound quantity Unity 1 Agar 190 g 2 Soy Protein 200 g 3 Yeast 200 g 4 Milk powder 140 g 5 Sorbic acid 9 g 6 Ascorbic acid 18 g 7 Methyl para benzene 15 g 8 Vitamin complex 30 g 9 Formalin 18 mm 10 Tetracycline 1 Pill 11 Rice 250 G 12 Beans 200 G