Insecticide Resistance Development and Detoxification Enzyme Activities of Spodoptera litura (Fabricius) in Soybean from Kumaon Himalayas

Spodoptera litura is one of the most important polyphagous lepidopteran pests in agriculture and belongs to the Noctuidae family (Wang et al., 2018; EFSA PLH Panel, 2018). This pest has enormous economic importance for field and horticultural crops (Murthy et al., 2006) and is widespread throughout tropical and temperate Asia (Kranz et al., 1977). Severe outbreaks of S. litura have been reported in Kota (Rajasthan) and Marathwada and Vidarbha (Maharashtra) in India, causing total damage of about US$ 4.5 crores and US$22.5 crores, respectively (CROPSAP, 2012). Soybean, which is an important crop of India and the country ranks fifth in world soybean production (SOPA, 2020), suffered about 24.7% damage from S. litura (Higuchi et al., 1991). Uttarakhand is a Himalayan state located in the north-western part of India (Sati, 2020). S. litura is one of the major defoliator moths that occur in different regions of Uttarakhand and cause significant crop losses. Singh and Sachan (1992) reported that defoliating moths play an important role at the pod stage and thereafter in soybean. Several insecticides were recommended for use against this pest, which exerted high selection pressure on populations of S. litura. This selection pressure was responsible for the development of resistance of S. litura to many insecticides (Shi et al., 2019). In the 2017 IRAC Newsletter, it was documented that S. litura was ranked 7^th among top 20 arthropods in terms of number of resistance cases. Resistance to insecticides is a complex process and may result mainly from four mechanisms: i) increased metabolism to non-toxic products, ii) decreased sensitivity of the target site, iii) decreased rate of penetration of the insecticide, iv) increased rate of excretion of the insecticide (Sakine, 2012). The simplest and most conventional detection of resistance is dose-mortality experiments conducted in the laboratory under controlled conditions (Brown, 1976). Another approach is the biochemical mechanism, which, when used in addition to basic toxicity studies, gives the best results. Resistance to insecticides has been found to be essentially due to two mechanisms, i.e., enhanced enzymatic detoxification of an insecticide or reduced sensitivity of a target enzyme to inhibition by the insecticide; out of the four mechanisms discussed (Brown, 1987). The farmers from locations surveyed were using insecticides such as Profenofos 40%+Cypermethrin 4% EC, Indoxacarb 14.5% SC, Chlorantraniliprole 18.5% SC, Carbofuran 3G, Chlorpyriphos 50%+ Cypermethrin 5% EC, Fipronil 3.5%+Chlorpyriphos 35% etc. which were provided by government agencies involved in agricultural work as per information provided by farmers themselves. Therefore, the present study investigated the resistance level of five different insecticides and five S. litura field populations collected during 2019 and 2020 from soybean fields of Kumaon, Himalayas. The results of this study can help in developing effective and timely management practises.

Field populations of S. litura egg masses and larvae in soybean fields were collected from four hill districts, namely Ramgarh (Nainital district), Bhujan (Almora district), Kameri, Bageshwar and Ganai (Pithoragarh district) in Kumaon, Uttarakhand in 2019 and 2020. Other crops observed during survey in farmers field were Soybean, Cabbage, Cauliflower, Colocacia, Capsicum and French bean. Larvae were reared on artificial diet (Ballal, 2003) and adults were fed on 10% honey solution under laboratory conditions (27±1°C and 65±5% relative humidity) with a photoperiod of 16:8 hours light: dark. Adults were kept in jars with butter paper attached for oviposition by females and reared to F1 generation. A susceptible population of S. litura was obtained from NBAIR, Bangaluru, for comparative studies. Based on the survey information on insecticides used by farmers in the selected regions, five insecticides were used in the study, namely indoxacarb 14.5% SC, chlorantraniliprole 18.5% Sc, fipronil 5% SC, chlorpyriphos 50%+cypermethrin 5% EC and profenofos 40%+cypermethrin 4% EC. The bioassay method of IRAC Method No. 7 (IRAC, 2010) was performed on third instar larvae of S. litura in Pulse Entomology Lab, Deptt of Entomology, College of Agriculture, GBPUAandT, Pantnagar. The insecticide solution was prepared from a 1% stock solution by serial dilution. Fresh castor leaves of uniform size i.e., 5 x 5 cm were taken and dipped in the insecticide solution for 10 seconds. Excess liquid was drained off and the leaves were then air dried for half an hour. Ten third instar larvae from the F1 progeny (after being starved for 6 hours) were transferred to each petri dish and mortality was monitored at 24, 48 and 72 hours post exposure. Tests were conducted at a controlled temperature of 27± 1°C, 65±5% relative humidity and a 16:8 L:D photoperiod. To investigate the potential of the detoxification enzymes, biochemical analysis was performed to test the total protein and estimate the activity of Carboxylesterase (CarE) (Devonshire,1977 and Van, 1962); Acetyl choline esterase (AChE) (Kranthi, 2005); Mono-oxygenase (P450) (Kranthi, 2005) and Glutathione-S-transferase (GST) (Van,1962 and Booth et al., 1961). Polo Suite Leora Software LLC was used to estimate the LC₅₀ values of the chemical bioassays and the specific activities of the biochemical studies were analysed using analysis of variance (ANOVA).

The survey to study the distribution and collection of S. litura was conducted in 2019 and 2020 in different districts of Kumaon region of Uttarakhand (Table 1, Fig 1). Visual estimation was used to determine the infestation level. The scales for determining infestation were given by Vennila et al., (2010) and used by Singh and Gandhi (2012) in their study on S. littoralis on cotton, cabbage, radish and other insect pests. Four scales were given for infestation: no insect/scares appearance (scale 0); scattered appearance of few (scale 1); severe incidence on only one branch (scale 2); severe incidence on more than one branch (scale 3) and complete severe incidence (scale 4). The study found that severe to complete severe incidence of the pest was observed in the regions studied. The S. litura samples were collected from different locations and brought to the laboratory for further rearing of the F1 generation, where bioassay tests were carried out (Table 2). Insecticide resistance results according to insecticide used and locations visited were classified according to Shen et al., (1991) insecticide resistance level classification, i.e., RR <3.0 (susceptible), 3.0<RR<5.0 (Decreased Susceptibility); 5.0<RR<10.0 (Low level of resistance); 10.0<RR<40.0 (Moderate level of resistance); 40.0<RR<160.0 (High level of resistance) and RR>160.0 (Extremely high level of resistance).
 

 

 

       
S. litura populations tested with indoxacarb 14.5% SC were found to be susceptible for Bhujan and Pithoragarh populations; low level of resistance was observed for Ramgarh; moderate level for Bageshwar. Wang et al., (2018) also reported low to moderate level of resistance between 2-31-fold of S. litura population in Sichuan, China, to indoxacarb 14.5% SC from 2014-2016. Chlorantraniliprole 18.5% SC showed moderate to high resistance at almost all sites. Muthuswamy et al., (2014) reported a resistance ratio of 80.07 with respect to the susceptible NBAIR population, which is in insecticide resistance class 4. The bioassay results showed that Fipronil 5% SC showed resistance at almost all sites, from low to very high levels of resistance. Ahmad and Mehmood (2015) found that Pakistani populations developed moderate resistance every 8 years, increasing from 5.5-5.6-fold in 1998 to 28-35-fold in 2006. Ahmad et al., (2008) also reported that 22-fold resistance was observed to Fipronil 5% SC and this high level of resistance was explained by a multiple resistance mechanism. For combination insecticides i.e., profenofos 40%+cypermethrin 4% EC and chlorpyriphos 50%+ cypermethrin 5% EC, almost similar results were observed with both insecticides at all locations except Bhujan and the possible reason could be the selection pressure of profenofos 40%+cypermethrin 4% EC since the farmers used only this insecticide in their fields. It was also observed that the population of Ramgarh showed sensitivity to the combination of insecticides, which may have been due to the strict control and regulations of some private companies with which the farmers collaborate for organic crop production in the area as mentioned by farmers in the area and due to the low relative humidity (RH) of the sampling site, as RH is an important factor determining the life span of S. litura (Kumar et al., 2013; Khan and Talukder, 2017). Both the profenofos 40%+cypermethrin 4% EC and chlorpyriphos 50%+cypermethrin 5% EC have an OP to pyrethroid ratio of 1:10 and have good resistance, which Ahmadi (2009), El-Guindy et al., (1983), Goebel and Jacquemard (1990) and Forrester et al., (1993) reported that profenofos and chlorpyrifos antagonize cypermethrin in a ratio of 1:10.
       
Populations from different districts of Uttarakhand were subjected to biochemical analysis to identify their levels of detoxification enzymes compared to susceptible populations from NBAIR, Bangaluru (Table 3 and 4). Almora Bhujaan population showed the highest specific activity for both acetylcholinesterase enzyme (6.770±0.140 nmol/min/ml enzyme) and monooxygenase P450 enzymes (2.780±0.630 nmol/min/ml enzyme) and resistance ratio 8 .63 and 111.20 were found in the reference laboratory population, respectively. For carboxylesterase enzyme, the highest specific activity was observed in the population of Kameri, Bageshwar, i.e., the formation of 0.505±0.035 µmoles 1-naphthol/min/mg protein and RR (resistance ratio) of 33.67; For glutathione-S-transferase enzyme, the highest specific activity was observed in Ganai, Pithoragarh population ie. 1.910±0.050 µmoles /min/mg protein with a RR of 23.87.
 

 

       
This study was conducted to investigate the level of infection and resistance to five different pesticides preferred by farmers in the region in four different areas of the Kumaon hills, Uttarakhand. The level of S. litura infestation was found to be on a scale of 3 and 4, meaning that it ranged from severe presence in more than one branch to full severe presence. According to Chattopadhyay et al., (2019) outbreak of S. litura was observed at temperature between 21-27°C and RH above 90%. The infestation scale was related with temperature, humidity and availability of abundant host since the favourable conditions generated due to these factors are important for the growth and development of S. litura (Joshi et al., 2022, Fand et al., 2015). The data of previous and collection month showed that Ramgarh and Bhujaan had a constant favourable climatic condition in addition to abundance of host. Also, the chemical bioassay studies showed that the population is resistant against some of the insecticides used implying better survival of insect population in region, in addition to favourable environmental factors. It was observed in the studies that the resistance in Fipronil 5% SC and Chlorantraniliprole 18.5% SC could not be directly correlated with activity of any detoxification enzymes. Arain et al., (2018) reported that detoxification of Fipronil 5% SC is not strongly related to the activity of these detoxification enzymes, as well as Su et al., (2012) mentioned similar results for 18.5% SC resistance to chlorantranilprole and suggested that another mechanism may be responsible for detoxification of this insecticide. The level of resistance to the combination insecticides can be related to high activity of esterase enzymes i.e. AChE and CarE, but no such direct relationship with activity was observed for GST and P450 enzymes, also shown by Muthusamy et al., (2011) in their studies on insecticide detoxification mechanisms in S. litura. Similar results were obtained comparing Indoxacarb 14.5%SC resistance with detoxification enzyme activity, as no correlation was observed between enzyme activity and insecticide resistance, suggesting a possible role for other detoxification mechanisms (Shi et al., 2019). Enzymatic activity indicated that overproduction of detoxification enzymes may be one of the main factors contributing to insecticide resistance in the studied S. litura populations (Despres et al., 2007).

Soybean is an important crop in Uttarakhand and is important pulse in the diet of people from hills of Kumoan. Development of insecticide resistance in S. litura which is one of the major insect-pests of soybean has already been reported from different parts of world. This study found that insecticide resistance could become a major concern for hill farmers in Uttarakhand, India. Multiple mechanisms are responsible for the development of insecticide resistance in insects, which requires further investigation, making it a promising area of   research for developing integrated insect control strategies.

We are thankful to G B Pant University of Agriculture and Technology, Pantnagar for providing all the support to conduct survey and lab research work.

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