Synergistic Toxicity of Selected Pesticides Combined with Serratia marcescens against Odontotermes formosanus (Isoptera: Termitidae) Workers1
Abstract
The termite, Odontotermes formosanus (Shiraki) (Isoptera: Termitidae), is an important pest in China. In this study, 7.75 × 108 CFU/mL of the bacterium Serratia marcescens Bizio SM1 (LT50 = 74.26 h) was combined with 1 mg/mL of RH-5849 (LT50 = 54.98 h), 1 mg/mL of buprofezin (LT50 = 92.50 h), 0.02 mg/mL of emamectin benzoate (LT50 = 105.21 h), 0.01 mg/mL of indoxacarb (LT50 = 64.89 h), and 0.01 mg/mL of ivermectin (LT50 = 74.66 h), respectively, at the volume ratios of 9:1, 5:1, 1:1, 1:5, and 1:9. Through the analysis of the bioassay results, it was found that when S. marcescens was combined with RH-5849 at the ratios (v/v) of 5:1, 1:1, 1:5 and 1:9 respectively, the co-toxicity coefficients (CTC) were 155.46, 201.41, 234.37, and 148.50, respectively. Similarly, combinations of S. marcescens with buprofezin at the ratios (v/v) of 9:1, 5:1, and 1:1, the CTCs were 135.00, 159.57, and 181.38, respectively, showing synergistic toxicity (CTC > 120) against O. formosanus. Synergistic activity also occurred when S. marcescens was combined with emamectin benzoate at the ratios (v/v) of 9:1, 5:1, 1:1, 1:5, and 1:9 with resulting CTCs of 181.43, 161.47, 178.52, 172.50, and 221.69, when S. marcescens was combined with indoxacarb at the ratios (v/v) of 5:1, 1:1, 1:5, and 1:9 yielding CTCs of 123.81, 158.67, 154.91, and 168.51, and when S. marcescens was combined with ivermectin at the ratios (v/v) of 5:1, 1:5, and 1:9 with CTCs of 123.92, 128.19, and 188.05. These results provide a theoretical basis for management of O. formosanus.
Termites are eusocial insects that usually feed on substances composed of cellulose, thereby causing considerable damage to buildings, agricultural and forestry products, standing trees and forests, dams, etc. and inflicting significant losses to human society (Chiu et al. 2022, Su and Scheffrahn 2000, Wang et al. 2018). Therefore, the search for safe and highly effective agents is a direction to be considered in termite management. Given the limitations of conventional chemical strategies, including resistance development and ecological risks, developing sustainable biocontrol solutions with entomopathogenic microorganisms is a critical priority in termite management (Clercq et al. 2011). Serratia marcescens Bizio is a Gram-negative bacterium that is commonly found in both laboratory-reared and field-collected insects and has pathogenic effects on many insects, including those of Orthoptera, Homoptera, and Lepidoptera (Aggarwal et al. 2017, Nehme et al. 2007, Niu et al. 2015). Several research studies have indicated that S. marcescens holds great promise for application in the biological control of pests (Bidari et al. 2017, Jupatanakul et al. 2020, Zhao et al. 2020). Our previous research also demonstrated that S. marcescens (SM1) is effective against termites (Fu et al. 2021, Wang et al. 2024). Therefore, applying S. marcescens for pest control shows great potential.
In addition to being directly used as biological agents for pest control, insect pathogens can also serve as biological synergists for chemical pesticides. The distinct advantages of insect pathogens and chemical pesticides make their combined application a great method to address many pest problems (Paula et al. 2011). The combined application of insect pathogens and chemical pesticides can increase the toxicity to pests while reducing the application doses of each other (Subbanna et al. 2019). For example, in the research on the combined application of the fungus Isaria fumosorosea (Wise) Kepler, Shrestha & Spatafora (Hypocreales: Cordycipitaceae) and 4 chemical pesticides (spirotetramat, acetamiprid, imidacloprid, and thiamethoxam) for the control of Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae), a substantial synergistic effect was found. When the conidia were applied together with the chemical pesticides, the insecticidal effect was enhanced (Zou et al. 2014). Narkhede et al. (2017) discovered that the combined use of Bacillus thuringiensis Berliner BTSV2 and temephos led to a reduction in the median lethal dose (LD50) for individual pesticide application. Moreover, when compared to the use of individual pesticides, the larvicidal efficacy was enhanced (Narkhede et al. 2017). These studies demonstrated the potential of innovative combined biological pesticides. The combination of entomopathogenic microorganisms and chemical pesticides may be a valuable pest control strategy and also provide a feasible approach to reducing the amount of chemical pesticides used in pest management (Wang et al. 2013).
Odontotermes formosanus (Shiraki) (Isoptera: Termitidae), being a significant pest, not only can damage the bark and roots of plants but also poses a serious threat to dams (Huang et al. 2006). Fu et al. (2021) isolated the S. marcescens strain SM1 from O. formosanus and discovered the components that can kill termites. Based on these findings, we conducted a study on the combination of S. marcescens SM1 and various pesticides to determine whether the combination of S. marcescens SM1 with 5 pesticides having different mechanisms of action, namely 1,2-dibenzoyl-1-tert-butylhydrazine (RH-5849), buprofezin, emamectin benzoate, indoxacarb, and ivermectin, would exhibit antagonistic, additive, or synergistic toxicity against O. formosanus. The combined bio-preparations with synergistic toxicity against O. formosanus obtained from this study will provide new evidences for the control of pests by the combined agents of insect pathogens and new pesticides. Meanwhile, it also offers a new theoretical basis for the research and development of combined pesticides against O. formosanus.
Materials and Methods
Source of insects
Specimens of O. formosanus were collected from Nanjing, Jiangsu Province. The O. formosanus colonies were placed into acrylic devices that have been divided into colony area and feeding area. The termites were then reared under the conditions of room temperature at 25 ± 1°C, 80 ± 5% relative humidity (RH), and 24 h darkness.
Sources of entomopathogen and pesticides
The S. marcescens strain SM1. isolated from termite cadavers displaying reddish post-mortem discoloration, was purified and cryopreserved at −80°C. The RH-5849 technical grade active ingredient was purchased from Jiangsu Greenscie Chemical Co., Ltd. (Jiangsu. China); 98% buprofezin (BUP) technical grade active ingredient was purchased from Shandong Huayang Pesticide Chemical Industry Group Co., Ltd. (Shandong, China), 74.2% emamectin benzoate (EMB) technical grade active ingredient was purchased from Nanjing Red Sun Co., Ltd. (Karachi, Pakistan); 93% indoxacarb (IND) technical grade active ingredient was purchased from Jiangsu Jiannong Plant Protection Co., Ltd. (Jiangsu, China), and; ivermectin (IVE) was purchased from Zhongnong Warwick Bio-Pharmaceutical (Hubei, China) Co., Ltd.
Culturing S. marcescens SM1
The cryopreserved S. marcescens SM1 strain stock culture was retrieved from the −80°C ultra-low temperature freezer and thawed. One μL was added to 50 mL of the fermentation medium (1 L: peptone 10 g, soybean oil 30 g, NaCl 2 g, and K2HPO4 2 g; pH = 7.5) that had been sterilized by autoclaving at 121°C for 25 min continuously. This was placed on a shaker (30°C, 180–200 rpm) for 36 h. Shaking of the culture was halted when the OD600 = 0.6. When the bacterial suspension was used, the bacterial suspension was diluted to 7.75 × 108 CFU/mL for use in the assays.
Preparation of compounding agents
Stock solutions of RH-5849, buprofezin, emamectin benzoate, and indoxacarb were prepared at a concentration of 1 mg/mL using acetone. The stock solutions of RH-5849 and buprofezin were directly utilized as experimental concentrations. Subsequently, the 1 mg/mL indoxacarb stock solution was diluted 100-fold with acetone to yield a 0.01-mg/mL solution for future use, and the 1 mg/mL EMP stock solution was diluted 50-fold with acetone to create a 0.02-mg/mL solution for standby. Additionally, the ivermectin was diluted with acetone to similarly obtain a 0.01-mg/mL solution for standby. All the prepared solutions were then stored at 4°C. Subsequently, 7.75 × 108 CFU/mL S. marcescens SM1 was homogenized with 1 mg/mL RH-5849, 1 mg/mL buprofezin, 0.02 mg/mL emamectin benzoate, 0.01 mg/mL indoxacarb, and 0.01 mg/mL ivermectin, respectively, to formulate combined agents with volume ratios (v/v) of 9:1, 5:1, 1:1, 1:5, and 1:9.
Bioassays
A 7-cm diameter qualitative filter paper was placed flat in a suitably sized Petri dish to which 600 μL of the combined agent to be tested was evenly deposited from the outer edge to the inner part of the filter paper. The treated filter paper was then spread to ensure the paper fit the dish bottom tightly without gaps. After being allowed to dry slightly, the Petri dish was covered and set aside for use. Thirty healthy O. formosanus workers of similar instars were placed in each Petri dish. The experiment was conducted under dark conditions at 25 ± 1°C and 80 ± 5% RH. Observations were made once every 12 h. Termites demonstrating an inability to right themselves and showing no locomotory response to gentle tactile stimulation using a soft-bristled brush were classed as deceased. The dead termites were then removed, counted, and recorded. Throughout the observations, attention was given to adding water (using clear water as the water source) to the filter paper to keep it moist until the end of the experiment. The combined agents were treated according to the above 5 combination ratios with 4 replicates per treatment, with acetone as the control.
Statistical methods
The mortality of O. formosanus was corrected using Abbott’s formula (Abbott 1925). All data were processed using the DPS Data Processing System (Tang and Zhang 2013) to calculate the median lethal time (LT50). The combined toxic effect of S. marcescens SM1 mixed with different agents against O. formosanus was analyzed using the co-toxicity coefficient (CTC) (Sun and Johnson 1960), where Toxicity Index (TI) of the standard pesticides is 100, Toxicity Index (TI) = (LT50 of the standard pesticide/LT50 of the tested single agent) × 100, Actual Toxicity Index (ATI) = (LT50 of the standard pesticide/LT50 of the mixture) × 100, Theoretical Toxicity Index (TTI) = (TI of single-agent A × percentage of agent A in the mixture + TI of single-agent B × percentage of agent B in the mixture) × 100 and CTC = (ATI of the mixture/TTI of the mixture) × 100. The resultant CTC value was used to indicate antagonistic toxicity (CTC < 80), additive toxicity (CTC = 80–120), or synergistic toxicity (CTC > 120) as per Sun and Johnson (1960).
Results
Toxicity evaluation of S. marcescens SM1 + RH-5849 against O. formosanus
The LT50 of 7.75 × 108 CFU/mL S. marcescens SM1 against O. formosanus was 74.26 h, while that of 1 mg/mL RH-5849 was 54.98 h (Table 1). Their combinations at volume ratios (v/v) of 9:1, 5:1, 1:1, 1:5, and 1:9 resulted in LT50 values of 63.24 h, 45.13 h, 31.37 h, 24.52 h, and 38.01 h, respectively. LT50 values decreased from 63.24 h to 24.52 h as the RH-5849 proportion increased from 10% to 90%, then slightly rebounded to 38.01 h at the 1:9 ratio.
Using S. marcescens SM1 as the standard agent, the TI of RH-5849 was 135.07 (Table 2). Combining S. marcescens SM1 (7.75 × 108 CFU/mL) with RH-5849 (1 mg/mL) at volume ratios (v/v) of 9:1, 5:1, 1:1, 1:5, and 1:9 yielded an ATI of 117.43, 164.55, 236.72, 302.85, and 195.37, a TTI of 103.51, 105.84, 117.53, 129.22, and 131.56, and a CTC of 113.45, 155.46, 201.41, 234.37, and 148.50 against O. formosanus. All combinations, except the 9:1 ratio, demonstrated synergistic toxicity (e.g., CTC > 120), with the highest synergism observed at the 1:5 ratio (CTC = 234.37). The 9:1 ratio exhibited additive toxicity (CTC = 113.45).
Toxicity evaluation of S. marcescens SM1 + buprofezin (BUP) against O. formosanus
The LT50 of 7.75 × 108 CFU/mL S. marcescens SM1 against O. formosanus was 74.26 h, while that of 1 mg/mL buprofezin was 92.50 h (Table 3). Their combinations at volume ratios (v/v) of 9:1, 5:1, 1:1, 1:5, and 1:9 resulted in LT50 values of 56.12 h, 48.12 h, 45.42 h, 111.97 h, and 90.26 h, respectively.
Using S. marcescens SM1 as the standard agent, the TI of buprofezin was 80.28 (Table 4). Combining S. marcescens SM1 (7.75 × 108 CFU/mL) with buprofezin (1 mg/mL) at volume ratios (v/v) of 9:1, 5:1, 1:1, 1:5, and 1:9 yielded ATIs of 132.34, 154.32, 163.50, 66.32, and 82.27, TTIs of 98.03, 96.71, 90.14, 83.57, and 82.25, and CTCs of 135.00, 159.57, 181.38, 79.36, and 100.02 against O. formosanus. Three combinations (e.g., 9:1, 5:1, 1:1) showed synergism (CTC = 135.00–181.38 >120), while the 1:5 ratio exhibited antagonism (CTC = 79.36 < 80) and the 1:9 ratio had additive toxicity (80 ≤ CTC <120) against O. formosanus. The highest synergistic effect was observed at the 1:1 ratio (CTC = 181.38).
Toxicity evaluation of S. marcescens SM1 + emamectin benzoate (EMB) against O. formosanus
The LT50 of 7.75 × 108 CFU/mL S. marcescens SM1 against O. formosanus was 74.26 h, and that of 0.02 mg/mL emamectin benzoate was 105.21 h (Table 5). Combination at volume ratios (v/v) of 9:1, 5:1, 1:1, 1:5, and 1:9 yielded LT50 values of 42.17 h, 48.36 h, 48.77 h, 57.03 h, and 45.56 h, respectively, demonstrating significant synergy at the higher S. marcescens SM1 ratios.
Using S. marcescens SM1 as the standard agent, the TI of emamectin benzoate was 70.58, while the S. marcescens SM1 (7.75 × 108 CFU/mL) combined with emamectin benzoate (0.02 mg/mL) at volume ratios (v/v) of 9:1, 5:1, 1:1, 1:5, and 1:9 exhibited ATIs of 176.10, 153.56, 152.27, 130.21, and 162.99 respectively, and TTIs of 97.06, 95.10, 85.29, 75.49, and 73.52, and CTCs of 181.43, 161.47, 178.52, 172.50, and 221.69, respectively. All combinations demonstrated synergistic toxicity against O. formosanus as CTC values exceeded 120, with the 1:9 ratio showing the highest level of synergism (CTC = 221.69) (Table 6).
Toxicity evaluation of S. marcescens SM1 + indoxacarb (IND) against O. formosanus
The 7.75 × 108 CFU/mL concentration of S. marcescens SM1 resulted in an LT50 of 74.26 h against O. formosanus, while 0.01 mg/mL indoxacarb alone had an LT50 of 64.89 h (Table 7). When S. marcescens SM1 (7.75 × 108 CFU/mL) was combined with indoxacarb (0.01 mg/mL) at volume ratios (v/v) of 9:1, 5:1, 1:1, 1:5, and 1:9, the LT50 values against O. formosanus were 82.57 h, 58.57 h, 43.65 h, 42.79 h, and 39.00 h, respectively.
Using S. marcescens SM1 as the standard agent, indoxacarb had a TI of 114.44 (Table 8). When S. marcescens SM1 (7.75 × 108 CFU/mL) was combined with indoxacarb (0.01 mg/mL) at volume ratios (v/v) of 9:1, 5:1, 1:1, 1:5, and 1:9, the ATIs of the mixtures were 89.94, 126.79, 170.13, 173.55, and 190.41, respectively, with TTIs of 101.44, 102.41, 107.22, 112.03, and 113.00, and CTCs were 88.66, 123.81, 158.67, 154.91, and 168.51, respectively. For the 9:1 ratio (CTC = 88.66 < 120), the combination exhibited additive toxicity against O. formosanus. For the ratios of 5:1, 1:1, 1:5, and 1:9 (CTC = 123.81–168.51 > 120), the combinations displayed synergistic toxicity against O. formosanus.
Toxicity evaluation of S. marcescens SM1 + ivermectin (IVE) against O. formosanus
The 7.75 × 108 CFU/mL concentration of S. marcescens SM1 resulted in an LT50 of 74.26 h against O. formosanus, while 0.01 mg/mL ivermectin alone had an LT50 of 74.66 h (Table 9). When S. marcescens SM1 (7.75 × 108 CFU/mL) was combined with ivermectin (0.01 mg/mL) at volume ratios (v/v) of 9:1, 5:1, 1:1, 1:5, and 1:9, the LT50 values against O. formosanus were 65.11 h, 59.98 h, 77.63 h, 58.19 h, and 39.68 h, respectively.
As per Table 10, with S. marcescens SM1 designated as the standard agent, ivermectin had exhibited a TI of 99.46. When S. marcescens SM1 (7.75 × 108 CFU/mL) and ivermectin (0.01 mg/mL) were combined at different volume ratios (v/v) of 9:1, 5:1, 1:1, 1:5, and 1:9, the ATIs of the mixtures were 114.05, 123.81, 95.66, 127.62, and 187.15, respectively, with TTIs of 99.95, 99.91, 99.73, 99.55, and 99.52, and CTCs were 114.11, 123.92, 95.92, 128.19, and 188.05, respectively. For the 9:1 and 1:1 ratio (CTC = 114.11 and 95.92, both < 120), the combination exhibited additive toxicity against O. formosanus. For the ratios of 5:1, 1:5, and 1:9 (CTC = 123.92–188.05 > 120), the combinations displayed synergistic toxicity against O. formosanus.
Discussion
Our bioassays showed that the combined agents of entomopathogenic microorganisms and chemical pesticides may have a potential as synergistic effects against target pests. Our results with S. marcescens and several pesticides corroborate the findings of similar studies. Feng et al. (2004) reported that the combined use of the oil formulation of the entomogenous fungus Beauveria bassiana (Balsamo Criv.) Vuillemin (Hypocreales: Clavicipitaceae) and imidacloprid for controlling the leafhopper Empoasca vitis (Gothe) (Hemiptera: Cicadellidae) yielded significant results, outperforming the application of either the fungus or imidacloprid alone. Shi and Feng (2006) used a combination of B. bassiana and pyridaben to control the citrus red mite, Panonychus citri (McGregor) (Acari: Tetranychidae). Their results indicated that, compared with the single agents, this fungus-pesticide combination could control the occurrence of P. citri more sustainably and effectively. The effect of S. marcescens S-JS1, either alone or combined with spirotetramat and thiamethoxam, against nymphs of Nilaparvata lugens Stal (Hemiptera: Delphacidae) exhibited synergistic or additive effects when compared to use of the bacterium or the pesticides along (Niu et al. 2018). Our experiment also demonstrated that the combined agents, in most proportions tested, exhibited a synergistic effect against O. formosanus compared to the effect of the single agents.
Chemical pesticides might suppress the physiological and immune activities of pests, diminishing their resistance to entomopathogenic microorganisms. Consequently, pests formerly not susceptible to entomopathogenic microorganisms could become susceptible, thus boosting the insecticidal efficiency. The combination of pesticides having different modes of action might also contribute to the synergistic effect of combined agents (Sandeep et al. 2022). Nevertheless, the specific mechanisms underlying the synergistic effect need further investigation.
Certainly, an antagonistic or additive effect can also occur when insect pathogens are used in combination with chemical pesticides. In a study evaluating combined control of B. tabaci using B. bassiana and imidacloprid, James and Elzen (2001) observed and antagonistic effect. They noted that the fungus hindered the efficacy of imidacloprid. When used together, the response of B. tabaci was either less than or similar to that with imidacloprid alone. They hypothesized that B. bassiana might induce a behavioral response in the whiteflies, decreasing their feeding and intake of imidacloprid. Mantzoukas et al. (2020) also observed that the combined toxic effects of cannabidiol oil and 3 biopesticides against Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), Rhyzopertha dominica F. (Coleoptera: Bostrichidae), Prostephanus truncatus Horn (Coleoptera: Bostrichidae), and Trogoderma granarium Everts (Coleoptera: Bostrichidae) tended to be more additive. This finding is similar to our findings in that S. marcescens SM1 exhibited antagonistic and additive effects in some combination ratios with the 5 pesticides we tested, not showing synergistic toxicity in all ratios against O. formosanus.
In our experiment, two novel insect growth regulators (IGRs), namely RH-5849 and buprofezin, were tested. RH-5849 belongs to a class of non-steroidal ecdysteroid agonists. It forces insects to molt ahead of schedule and also plays a role in suppressing their feeding behavior, thereby disrupting the normal development of the affected insects (Bengochea et al. 2013, Bhagyalakshmi and Raja 2022). Buprofezin, an inhibitor of insect chitin biosynthesis, mainly hinders insect molting, resulting in developmental arrest and death (Pan et al. 2024). Their insecticidal effects are relatively slow but well-suited to the characteristics of termites like O. formosanus. Foraging O. formosanus termite workers can transport pesticides back to the nest, which they have either ingested or externally carried on their bodies. Through trophallaxis (Su and Scheffrahn 1996), grooming (Liu et al. 2019), and other social behaviors, the pesticides are spread within the nest. Once termites within the nest accumulate pesticidal toxicants to lethal concentrations, complete eradication of the entire colony can be achieved.
Our experiment involved combining S. marcescens SM1 with 2 IGRs, RH-5849 and buprofezin. We found that, at certain ratios, the combined formulations showed significant synergistic toxicity against O. formosanus. In the study by Sadiq et al. (2022), it was also discovered that the combination of B. bassiana and IGRs had synergistic toxicity against the larvae of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) compared with the single-agent treatment groups, significantly boosting larval mortality. Bilal et al. (2017) also found that the combinations of B. bassiana and different IGRs exhibited synergistic effects against both T. castaneum and T. granarium. Hadi et al. (2013) reported similar results as well. These results indicate that the combined use of entomopathogenic microorganisms and IGRs holds great potential for termite control.
Contributor Notes
Jiangsu Hydraulic Research Institute, Nanjing 210017, China.
Water Resources Department of Jiangsu Province, Nanjing 210029, China.