Field persistence of certain new insecticides and their efficacy against black cutworm, Agrotis ipsilon (Hufnagel)

Chemical control is used as a rapid and reliable method for insect control. Still, there is an ongoing need to replace older conventional insecticides with new insecticides to maintain efficacy and environmental protection. Emamectin benzoate, indoxacarb and chlorantraniliprole are broad-spectrum insecticides with a novel mode of actions. The effects of these compounds on some biological aspects of the black cutworm, Agrotis ipsilon, and their field persistence residues were estimated. The results showed that egg hatch was affected by high concentrations (50 and 100 ppm) of the tested compounds. Larvae that hatched from treated eggs were significantly affected at concentrations of 25 ppm and higher. 1st-instar larvae were the most susceptible developmental stage. There was strong suppression of adult formation; 65 and 91% at 25 and 50 mg L−1, respectively. Profoundly affected larvae died before pupation; slightly affected ones reached pupation 2–4 days later, were smaller than larvae in the untreated control, and were sometimes unable to develop into normal adults. Comparatively high concentrations (50 and 100 mg L−1) of the test compounds were necessary to affect adults by ingestion. According to the results, the tested insecticides could be arranged according to their potency descendingly as follows: emamectin benzoate, indoxacarb, and chlorantraniliprole, respectively. Based on the field application, emamectin benzoate proved to be the most effective in initial and residual activity, causing 100% mortality while indoxacarb was least effective. Data also indicated that emamectin benzoate had the longest half-life (Lt50) while indoxacarb recorded the shortest one. The results obtained in this study indicate that emamectin benzoate, indoxacarb and chlorantraniliprole are potent compounds for controlling A. ipsilon. Therefore, these compounds are promising materials that can be used as alternative components in integrated pest management programs to reduce as possible the harmful of using conventional insecticides under field conditions.


Background
The black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), is the most destructive pests against many economically important crops, including cotton. At present, broad-spectrum chemical control targeting this pest is the most widely practiced management tool. However, the intensive use of insecticides has led to the development of widespread and multiple forms of resistance. The aim of recent researches focuses on the development and valuation of various alternative control strategies to reduce dependence on conventional pesticides. Recently, there is an urgent need for effective new insecticides are for continued successful control of A. ipsilon in the future. Several insecticides with different modes of action Ismail Bull Natl Res Cent (2021) 45:17 (chlorantraniliprole, emamectin benzoate, and indoxacarb) were chosen for this study (Demkovich et al. 2018).
Chlorantraniliprole is a new anthranilic diamide insecticide, which effectively controls pest insects belonging to Lepidoptera, Coleoptera, Diptera, and Hemiptera, is effective against insects that have developed resistance against some conventional insecticides (Bentley et al. 2010). Anthranilic diamides selectively bind to ryanodine receptors in insect muscles resulting in an uncontrolled release of calcium from internal stores in the sarcoplasmic reticulum (Lahm et al. 2005;Cordova et al. 2006), causing impaired regulation of muscle concentration leading to feeding cessation, lethargy, paralysis, and death of target organisms. Anthranilic diamides have very low vertebrate toxicity due to a > 500-fold differential selectivity toward insects over mammalian ryanodine receptors (Dinter et al. 2008).
Emamectin benzoate is a novel semi-synthetic derivative of natural product abamectin in the Avermectin family. Avermectins, including emamectin benzoate, is effective against a broad spectrum of arthropod pests (Pienkowski and Mehring 1982). These materials act by interfering with the action of gamma-aminobutyric acid (GABA) (Wright 1984). It blocks post-synaptic potentials of the neuromuscular junction, leading to paralysis.
Indoxacarb represents a new class of insecticides Oxidiazines, with its stomach and contact (Wise et al. 2006). It blocks the movement of sodium ions into the nervous system, resulting in paralysis and death of the pest.
The objectives of this study were to elucidate the effect of chlorantraniliprole, emamectin benzoate, and indoxacarb on the biological aspect of A. ipsilon. The study aims also to determine the residues persistence period, Lt 50 (time required to give 50% mortality) under field conditions.

Insect rearing
Culture of A. ipsilon was reared on castor bean leaves in Department of Insect Population Toxicology, Central Agricultural Pesticides Laboratory, Agriculture Research Center, Dokki, Giza, Egypt, for several generations under reared chamber environments were (25 ± 2 °C and 65 ± 5% R.H) without any insecticidal contamination and light-dark period was 12 h. Larvae reared in a big glass container with sawdust in the bottom. For adult emergence and oviposition, pupae were put in a ventilated cage. Adults were fed on a 10% sucrose solution soaked in a piece of cotton; laid eggs were collected and kept in glass containers for hatching to be used for the study.

Immature bioassays
Emamectin benzoate, indoxacarb and chlorantraniliprole against A. ipsilon egg mass were determined. The upper layers of each egg mass (0-24 h old) were removed gently with a fine hairbrush. The lower layer in each egg mass was counted by the binocular. The counted egg samples were dipped (5 s) in different aqueous concentrations of the tested formulations, or distilled water as a control. After drying for 2 h at room temperature, the treated eggs were stored in Petri dishes (9 cm in diameter). Three days later, untreated castor bean leaves were added to the Petri dishes for larvae to feeding. Egg hatchability % and larval survival were monitored daily, and the mortality percentages were calculated. Results are based on four replicates of each treatment.
In other assays, 1st-, 3rd-and 5th-instars larvae (10 per each replicate) were exposed to castor bean leaves (Ricinus communis L.) treated with a series of aqueous concentrations of the test formulations, or with distilled water as a control. Leaves were dipped for 10 s. in different concentrations of tested compounds, held vertically to allow the excess solution to drip off and dried for 2 h, then offered to larvae for 42 h. After these time intervals, the treated leaves were replaced by untreated ones. Ten 1st-instar larvae were kept in Petri dishes, 3rd-and 5thinstars were held in ventilated wood boxes. The boxes contained sawdust to reduce moisture. Larval mortality recorded after 24 h. Larvae were weighed before exposure to treated leaves and again six days (3rd-instar) or (5th-instar) after exposure. Control 3rd-and 5th-instar larvae, fed untreated leaves, were weighed after similar time intervals. Weight gains of the 3rd-and 5th-instar larvae were determined 24 h, after feeding on treated leaves. Pupation and adult formation of 5th-instars were evaluated after the start of the assay.

Adult bioassays
Effects of ingestion of the tested formulations by adults were studied by allowing adults to fed on a 10% sucrose solution containing 50 or 100 mg L −1 of each test compound, or 10% sucrose solution as a control. Male and female moths were kept in a glass jar containing five individuals each. Mortality was checked daily for five days. Solutions were changed every second day to prevent fungal growth. Results are based on four replicates.

Table 1 Effect of the tested formulations on the % egg-unhatch and larval survival of A. ipsilon
Means followed by the same letter in the same column are not significantly different according to Tukey's multiple range test at P < 0.05

Field experiment and sampling
Application of the tested insecticides was conducted during the cotton-summer season in El-Dakahlia Governorate, Egypt, at Aga district, whereas an area of 2000 m 2 was selected to be sown on 4th April 2019 with cottonseed var. "Giza 86. " The experiments were laid out in Randomized Complete Block Design (RCBD) (four replications). This area was divided into plots size each of 50 m 2 . Four treatments, the three tested insecticides and the check were designed in this area. Application of tested insecticides was done on 26th of June 2019 under field conditions. Irrigation water was used in diluting of the tested insecticides at their field recommended rates/200 L per feddan. Knapsack sprayer (CP3) equipped with one nozzle was used. Unplanted belts (3 m width) were left as barriers between plots to avoid contamination with drifts. Plots received all good recommended agricultural practices without any insecticidal treatments applied during the season. Samples of treated cotton leaves were collected randomly from each treatment immediately after one h from spray (zero time) and then 3, 6, 9, 12 and 14 days' post-spray and transferred directly to the laboratory for feeding A. ipsilon by treated leaves with tested insecticides, (100 larvae/treatment/ interval) for one day of each interval. Alive larvae were fed for another two days on untreated leaves. Cumulative mortalities were calculated at the end of each interval (three days) and corrected according to Abbott's formula (1925). The cumulative mortalities of the first intervals (samples collected after spraying directly) was considered as initial kill, while the total mean of the cumulative mortality of the other intervals (3, 6, 9, 12 and 14 days from spraying) were considered as residual effects.

Statistical analysis
Data were corrected for control mortality according to Abbott's formula (Abbott 1925). Also, mortality values were analyzed by Probit analysis (LPD line) to determine the LC values and slope for each compound according to a method adopted by Finney (1971). The results were analyzed using one-way ANOVA, significant differences between treatments were determined using Tukey's HSD test in JMP 11.1.1. (SAS Institute Inc. 2013, Cary, NC, USA).

Effect on eggs and young larvae
High concentrations of each compound were required to affect egg-hatch of A. ipsilon,

Susceptibility of A. ipsilon larvae to tested formulations
First-, 3rd-and 5th-instar A. ipsilon larvae were fed on castor bean leaves treated with various concentrations of the test compounds. All larval instars were more susceptible to the tested compounds than the eggs, Table 2. According to LC 50 value, 1st-instars are the most susceptible. Increases in mortality of 3rd-instar larvae were seen even after replacing treated leaves with untreated ones; heavily affected larvae were not able to recover and died at a later stage. There is no larva survived with the highest concentrations. The susceptibility of the 5th-instar larvae was lower than that obtained with the 3rd-instar, Table 3.

Effects on weight gain
Larvae that fed on tested compounds-treated leaves weighed less than control larvae, Table 3, the higher the concentration of tested compounds, the lower the larval weight. The weight gains of 3rd-instars exposed to 50 mg L −1 was 96% of the untreated larval weight, while larvae treated with 10 mg L −1 reached only 15% of the untreated larval weight. Fifth-instar larvae exposed to 25 ppm reached 45% of the larval weight of the untreated control, and many of those exposed to 25 mg L −1 experienced weight loss. Larvae of both stages were unable to maintain normal growth when fed on leaves treated with 50 mg L −1 , and almost half of the individuals lost weight.

Effect on pupation and adult formation
Pupation and adult formation of 5th-instar larvae were determined after the trial started. There was no significant difference between control larvae and those exposed to 10 mg L −1 ; approximately 80% of the larvae became adults, Table 4. Suppression of adult formation of 65 and 91% occurred at concentrations of 25 mg L −1 and 50 mg L −1 , respectively.

Effect of ingestion on adults
The effect of tested compounds by ingestion on mortality of A. ipsilon adult was tested by exposing male and female adults to sucrose solutions containing different concentrations of the tested compounds. A significant effect was observed at concentrations of 50 and 100 mg L −1 resulting in 35 and 60% mortality after three days of feeding, and in 93 and 87% mortality after five days of feeding, Table 5.

Persistence of the tested formulations residues
Leaves were collected from cotton field plots sprayed with tested compounds and fed to 3rd-instar A. ipsilon. The initial effect of emamectin benzoate proved to be the most effective where caused 100% accumulative mortalities after 3 days from treatment compared with control while indoxacarb was the least effective (60%). Based on the general mean of residual activity of the tested compounds, emamectin benzoate was superior compound giving 49.013%, followed by chlorantraniliprole 42.12%. On the other hand, indoxacarb (22.488%) was the least effective. Concerning the persistence of the residues of the tested compounds 1.11 ± 0.18

Table 3 Effect of tested formulations on mortality and weight gain of two larval instars of A. ipsilon
Means followed by the same letter in the same column are not significantly different according to Tukey's multiple range test at P < 0.05

Discussion
Results obtained from the present study indicate that although the tested compounds have low ovicidal activity yet, it has the highest residual activity against the neonates after 24 h of hatching. It was observed that the larvae of A. ipsilon died very soon after treatment probably resulted from their eating the eggshell and contacting the compound at a very young stage. These results are in harmony with in previous studies, Pinela et al. (2000) stated that treatment of 0-24 h eggs at 10 mg a.i/ liter or above caused 100% mortality of newly emerged larvae after the first day of hatching. Ioriatti et al. (2009) reported that chlorantraniliprole has an ovicidal activity low.
According to the estimated LC 50 values, the 1st instar larvae reflected higher level of susceptibility towards all the tested compounds than the 3rd-and 5th-instars, respectively. The obtained results showed the toxicity increased with the increasing of the exposure time and decreased by increasing the larval instars. The present results are confirmed with the results of Lisa 2015; Ismail 2018; Rimpy and Verma 2018, where they reported that emamectin benzoate is the most potent compound followed by indoxacarb, imidacloprid, pyridalyl and chlorantraniliprole against both of A. ipsilon and S. littoralis.
The results explained that the three newly insecticides significantly reduced biological aspects where weight loss as a result of larvae feeding on castor bean leaves treated with tested insecticides compared with control. Highly affected larvae were unable to increase weight and died before pupation. Slightly affected larvae reached pupation two to four days later than larvae in the untreated control, but pupae were smaller and sometimes unable to develop into normal adults and sometimes pupae can't complete the molting process and died. Although adults were strongly affected five days after exposure to the compounds, these effects were very mild when compared to effects on the larval stages. The potency of three tested compounds was similar between the sexes of A. ipsilon adults. Similar studies have shown that chemicals can have adverse effects on biological parameters, female mating behavior and fecundity of target insects (Xu et al. 2016;Mokbel et al. 2017;Barrania 2019).
Concerning the residue of emamectin benzoate and chlorantraniliprole could be toxic to A. ipsilon larvae longer than 7 days with relatively slow activity, but indoxacarb showed low residual toxicity. The rate of the tested compounds residues dissipation in cotton leaves was decreased gradually 3, 6, 9, and 12 days' post-treatment, and no residues could be detected 14 days' post-treatment. The present results are in aline with Gupta et al.   56 Ismail Bull Natl Res Cent (2021) 45:17 (2009) reported that the half-life time of indoxacarb at 70 and 140 g/ha on okra fruit was 0.58 and 1.02 days, respectively, whereas Dake and Bhamare (2019) evidenced the descending relative order of efficacy of insecticides in days was found to be indoxacarb 0.05% (4.74 and 4.93) > chlorantraniliprole 0.005% (4.19 and 4.38) > fenpropathrin 0.01% (3.84 and 3.68) > emamectin benzoate 0.002% (3.00 and 3.21) > flubendiamide 0.007% (2.56 and 2.86) against the nymph of jassid on sunflower leaves receiving first and second application of insecticides, respectively. From the present results, these selected insecticides are good candidates for controlling black cutworm, A. ipsilon and can replace hazardous convention insecticides.

Conclusions
The present results concluded that although indoxacarb effective against A. ipsilon and significantly reduced biological aspects, it has the lowest persistence period under field conditions. Concerning emamectin benzoate, and chlorantraniliprole caused high toxicity and significantly reduced biological aspects. Also, these compounds had the longest persistence residues and high initial effects under field conditions. Therefore, these compounds with a new mode of action could be used in strategies for controlling A. ipsilon and as alternatives of conventional insecticides.