Assessing the efficacy of un and gamma-irradiated entomopathogenic nematode, Steinernema carpocapsae as an eco-friendly approach to control Agrotis ipsilon larvae

The black cutworm, Agrotis ipsilon (Hufnagel) is one of the most devastating agricultural pests that attack different crops worldwide. Entomopathogenic nematodes (EPNs) have been utilized in classical, conservation, and augmentative biological control programs. Consequently, this study was designed to estimate the efficacy of an unirradiated and gamma-irradiated entomopathogenic nematode, Steinernema carpocapsae (all), against A. ipsilon larvae as a safe control tool to reduce chemical environmental hazards. The virulence of different concentrations (10, 20, 30 and 40 IJs/ml) of unirradiated and gamma-irradiated (with 2 Gy) of S. carpocapsae was evaluated. Changes in peroxidase (Px) and polyphenyl oxidase (PPO) zymographic patterns were also investigated after larvae were treated with LC50 values for S. carpocapsae. The results revealed that larval mortality increased with increasing S. carpocapsae concentration and time after treatment. The 2 Gy gamma-irradiated S. carocapsae caused a greater pathogenic effect than the unirradiated S. carocapsae, where the LC50 values of 2 Gy gamma-irradiated S. carocapsae were 31.19 and 17.78 IJs/ml after 1 and 2 days, respectively, compared to 113.23 and 65.83 IJs/ml for unirradiated S. carocapsae. For the toxicity index, gamma irradiation at a dose of 2 Gy was selected and given an arbitrary 100 units and considered a standard treatment. Analysis of gel electrophoresis revealed 3 bands of Px and PPO in control larvae; treatment with unirradiated and irradiated S. carpocapsae caused the disappearance of the first band of both enzymes and lowered the intensity of the other 2 bands. The laboratory studies revealed that 2 Gy gamma-irradiated S. carpocapsae could be used as an eco-safe management tool against A. ipsilon larvae under field conditions.


Introduction
The black cutworm, Agrotis ipsilon (Hufnagel), the black cutworm, is a destructive pest not only in Egypt but also throughout the world; it is most common in places that are temperate or subtropical (Kononenko 2003). This noctuid insect is polyphagous and infects numerous crops (Abo El-Ghar et al. 1996). Vegetables, cucurbitaceous plants, and agricultural crops are all infested by the greasy cutworm. The most damaged crops are cotton, essential-oil cultures, maize, tobacco, sunflower, tomatoes, sugar beet, and potato.
The larval stage is the most devastating; it prefers to live in the ground and feed on the stem, cutting off the stem and sometimes dragging the plants into their burrows. The larvae can consume over 400 cm 2 of foliage during their development. According to the larval habitat, it is difficult to control it (Amin et al. 2019).
The traditional control tool for this pest is using chemical insecticides, but the widespread use of these pesticides has resulted in a residual effect in the plant that produces new resistant insects and negative impacts on nontarget organisms.
Entomopathogenic nematodes (EPNs) of the "families Steinernematidae and Heterorhabditidae", along with the symbiotic bacteria Xenorhabdus and Photorhabdus, have been employed as biocontrol agents of pest insects that are harmful to agriculture (Gaugler 2002). When these bacteria are discharged from the nematode gut, they quickly grow in the host haemolymph, and within 24 to 48 h, they produce 1 3 deadly bacteraemia. Deadly endoparasites of insects known as Steinernematidae and Heterorhabditidae have the ability to secrete poisons, proteases, and other insecticidal active ingredients that can have a lethal effect on infected host insects (Toubarro et al. 2009). The deadly actions of insecticidal active ingredients frequently involve altered enzyme activity in host insects (Grewal et al. 2005).
Nematodes, like other living things, can be activated by low levels of gamma radiation (Marples and Collis 2008). Earlier research suggested that 2 Gy gamma-irradiated EPNs could be used to control certain insect infections (Yussef 2006;Sayed 2011;Salem et al. 2014;Sayed et al. 2015Sayed et al. , 2018Sayed and Shairra 2017).
Insects have an extremely effective cellular and humoral immune system (Gillespie et al. 1997). Phagocytosis, encapsulation, and nodule formation are all aspects of cellular immunity (Lavine and Strand 2002). Humoral defenses refer to soluble effector molecules, including antimicrobial peptides (AMPs), complement-like proteins, enzymes, and products generated by complex proteolytic cascades (Blandin and Levashina 2004).
Biochemically, enzymes are present in many alternative forms with the same enzyme activity, called isoenzymes, such as peroxidases and polyphenole oxidase. Peroxidases (Px) play a significant role in a variety of metabolic processes (Cheng et al. 2008) and contribute to xenobiotic detoxification, innate immunity, the pathogenesis of inflammatory diseases, and hormone biosynthesis (Lubos et al. 2011). Moreover, Prakasha and Umesha (2016) reported that peroxidases play a role in enhancing the plant's defense against pathogens and start working within minutes after a bacterial attack. Polyphenole oxidase (PPO) catalyzes the oxidation of phenols to quinones (Vanitha et al. 2009) and was reported at high levels after plant infection with bacterial diseases (Ngadze et al. 2012).
The current study aimed to evaluate the vulnerability of A. ipsilon's 5 th larval instar to unirradiated and gammairradiated S. carpocapsae. Beyond furthering its impact on some isoenzyme activity (Px and PPO) of the A. ipsilon larvae.

Rearing of A. ipsilon
The black cutworm larvae were reared on castor leaves away from any insecticidal contamination in the Natural Control lab., Natural Products Research Department, National Centre for Radiation Research and Technology, Egyptian Atomic Energy Authority, Cairo, Egypt, under laboratory constant conditions of 24 ± 2 °C and 65 ± 5% relative humidity and a photoperiod of 16 L:8 D. The newly hatched larvae were kept in a clean glass jar and provided with castor oil leaves daily until the 3 rd instar larvae emerged. Then, they were transferred to larger, clean glass jars to prevent larval cannibalism. The bottom of each jar was covered with a thick layer of fine sawdust, and the usual rearing techniques were performed along with the developing larvae until pupation occurred. The developed pupae were transferred to a clean jar for adult emergence after pupation. The adult moths (males and females in a 7:5 ratio) were transferred to a glass jar and given a dietary supplement of a hanged piece of cotton wool soaked in 10% sugar solution (El-Shershaby 2010).

Nematode
Stinernema carpocapsae (all) was obtained from the Biological Control Department, Plant Protection Research Institute; Agricultural Research Centre (ARC), Giza, Egypt. S. carpocapsae was cultivated on the last larval instar of Galleria mellonella L. (Bedding and Akhurst 1975) at the Natural Control lab., Natural Products Research Department, National Centre for Radiation Research and Technology, Egyptian Atomic Energy Authority, Cairo, Egypt, under controlled conditions of 25 ± 2 °C. Twenty larvae were placed in a 15 cm petri dish filled with filter paper, and then 2 ml of the infective juvenile suspension was sprayed. After the larvae were dead, they were placed in the collecting dishes, which consisted of filter paper on the glass watch placed in a larger and deeper Petri dish in which distilled water was added to reach the edge of the filter paper. The cadavers were placed in the middle of moist filter paper to migrate down into water. The juvenile suspension was collected daily by a special pipette and stored in glass jars containing distilled water in a refrigerator at 5 °C until it was used in the experiments.

Irradiation technique
The 3 rd infective juveniles (IJs) of the EPN species were gamma irradiated with 2 Gy (Sayed 2011) using the Gamma Cell Irradiation Unit located in the National Centre for Radiation Research and Technology, Egyptian Atomic Energy Authority. The dose rate used for the cesium unit (Cs 137 ) was 0.613 Rad/sec at the time of irradiation.

Virulence of unirradiated and irradiated S. carpocapsae on A. ipsilon larvae
The bioassay was carried out according to Woodering and Kaya (1988). Different concentrations of unirradiated and irradiated IJ nematode suspensions were prepared: 10, 20, 30 and 40 IJs/ml; the control consisted of the same volume of sterilized distilled water. Six larvae were placed in 100 cm 3 plastic cups, and 1 ml of each nematode concentration was sprayed. Each concentration and the control were replicated 5 times under controlled conditions of 25 ± 2 °C. The mortalities were counted daily, and the cumulative mortalities were calculated. The LC 50 values were calculated using Ldp-line software (copyrighted by Ehab Bakr, Plant Protection Research Institute, Agricultural Research Center, Giza, Egypt).

Isoenzyme electrophoresis
For isoenzyme extraction, 0.5 g of normal and treated larvae (with LC 50 values of unirradiated and irradiated S. carpocapsae) were homogenized in 1 ml of extraction buffer (10% glycerol). The extract was then transferred in to clean Eppendorf tubes and centrifuged at 10,000 rpm for 5 min. The supernatant was transferred to new, clean Eppendorf tubes and kept at -20 °C until used for electrophoretic analysis. Native-polyacrylamide gel electrophoresis (Native-PAGE) was conducted to identify isozyme variations among the studied larvae using two isozyme systems, according to Stegemann et al. (1985). The utilized isoezymes were peroxidase (Px) and polyphenol oxidase (PPO). A volume of 40 μl extract of each larvae group was mixed with 20 μl sucrose and 10 μ bromophenol blue, and then a volume of 50 μl from this mixture was applied to each well. The run was performed at 150 V until the bromophenol blue dye reached the separating gel, and then the voltage was increased to 200 V. The electrophoresis apparatus was placed inside a refrigerator during the running duration. After electrophoresis, the gels were stained according to their enzyme systems with the appropriate substrate and chemical solutions and then incubated at room temperature in the dark for complete staining (in most cases, incubation for approximately 1 to 2 h is enough).
For peroxidase, the gel was incubated at room temperature with 0.125 g of benzidine dihydrochloride, 2 ml of glacial acetic acid, 50 ml of dilute water, and 5 drops of hydrogen peroxide until bands appeared.
For polyphenyl oxidase, the staining substrate solution was prepared by 0.1 M phosphate buffer pH 6.5, 100 mg sulphonillic acid, and 200 mg cathecol in 2 ml acetone. The gel was placed into this solution and incubated at 30 °C for 30 min until bands appeared. Gels were photographed, scanned, and analyzed using the Gel Doc VILBER LOUR-MAT system.

Statistical analysis
The Minitab program was used to adjust and analyze the obtained results using ANOVA, followed by Tukey's pairwise comparisons test to examine the significant differences (P < 0.05) across the means of the treatments.

Results
The mortality rates of 5 th instar larvae of A. ipsilon by unirradiated S. carpocapsae at various time intervals were showed in Fig. 1. The data indicated a positive relationship between increased S. carpocapsae concentration and larval mortality, as well as exposure time (Fig. 1). The mortalities were 3.33, 6.67, 10, and 23.33% after 1 day and increased to 10, 16, 30, and 36.67% after 2 days of treatment with 10, 20, 30 and 40 IJs/ml, respectively. On the third day of the treatment with 10, 20, 30 and 40 IJs/ml, the percentages of mortality recorded the highest rate, being 16.67, 30, 40, and 56.67%, respectively.
The overall death rate showed a parallel correlation with increasing nematode concentration and exposure time (Fig. 2). The 5 th larval instar of A. ipsilon was highly susceptible to irradiated S. carpocapsae; larval mortalities were 100% after 2 and 3 days of treatment with irradiated 40 and 30 IJs/ml, respectively. After 1 day of treatment with 10, The data in Table 1 display the calculated LC 50 and LC 90 values of unirradiated and irradiated S. carpocapsae after 1 day and 2 days of treatment. The LC 50 values of unirradiated S. carpocapsae were 113.23 and 65.83 IJs/ml after 1 day and 2 days of treatment, respectively. For the irradiated S. carpocapsae, the corresponding values of 32.19 and 17.78 IJs/ml after 1 day and 2 days of treatment, respectively (Fig. 3). Generally, the results implied that A. ipsilon larvae were more susceptible to irradiated S. carpocapsae, with a toxicity index of 100, than unirradiated larvae.
The peroxidase (Px) zymogram of 5 th instar larvae of A. ipsilon either normal or treated with the LC 50 of unirradiated and irradiated S. carpocapsae presents in Table 2 and Fig. 4a show The analysis revealed 3 isomers, Px1, Px2, and Px3, with relative mobilities of 0.1, 0.3, and 0.45, respectively. Px1 appeared with moderate density only in control larvae and disappeared in the analysis of treated larvae. Px2 was detected in all samples, but in the control larvae, the density was high and lowered to moderate density in larvae treated with the LC 50 of unirradiated and irradiated S. carpocapsae. The density of the 3 rd band (Px3) was moderate in control larvae or larvae treated with the LC 50 of unirradiated S. carpocapsae. Px3 density was low in larvae treated with the LC 50 of irradiated S. carpocapsae.
Polyphenyl oxidase (PPO) isozyme patterns are presented in Table 3 and Fig. 4b. Three PPO isoezymes, PPO1, PPO2, and PPO3, were discovered in the 5 th instar larvae of A. ipsilon either normal or treated with the LC 50 of unirradiated and irradiated S. carpocapsae, and their relative mobility values were 0.1, 0.4, and 0.45, respectively. The PPO1 band was detected in control larvae only at a moderate density. Moreover, PPO2 and PPO3 showed high and moderate densities, respectively, in control larvae and decreased to low densities in larvae treated with the LC 50 of unirradiated and irradiated IJs.

Discussion
Stiner nema carpocapsae has demonstrated its effectiveness in controlling different insect pests (Bhairavi et al. 2021). The infectivity of the nematode depends on the species, stage, size, and habitat of the pest, and no one nematode species was the most infective for all insect species (Bedding et al. 1983).
Our results showed that there was a positive correlation between 5 th instar larvae of A. ipsilon mortality and S. carpocapsae concentration increases (unirradiated or irradiated) as well as the time of treatment increase. This agrees with Hassan et al. (2016), who stated that increasing the    According to the prior findings on LC 50 values, it was clear that the 2 Gy gamma-irradiated S. carpocapsae was more virulent against the 5 th instar larvae of A. ipsilon. The same finding of the increased pathogenicity of gammairradiated EPN compared to unirradiated EPN was previously reported by Yussef (2006), who stated that irradiated S. carpocapsae was more virulent against Callosobruchus maculatus (F.). Similarly, irradiated S. carpocapsae killed  G. mellonella, Corcera cephalonica (Stainton), and Ephestia kuehniella (Zeller) larvae faster than unirradiated larvae (Sayed 2011). Moreover, Sayed and Shairra (2017) evaluated the effectiveness of 2 Gy γ-irradiated S. scapterisci among Spodoptera littoralis (Boisd.), and they obtained the same result. Sayed et al. (2018) confirmed that the LC 50 of 2 Gy gamma-irradiated S. scapterisci was lower than that of unirradiated S. scapterisci when used against Bactrocera zonata (Saunders) larvae and pupae. This increase in pathogenicity could be explained by the fact that low doses of gamma radiation cause the symbiotic bacteria to grow more, increasing their toxin production. This was in accordance with Bashandy and El-sinary (2002), who stated that insect mortality rose from 20 to 66% when using gamma-irradiated Bacillus thuringiensis.
The gel electrophoresis analysis of peroxidase and polyphenyl oxidase patterns of 5 th instar larvae of A. ipsilon (control and treated with LC 50 of unirradiated and irradiated S. carpocapsae) revealed the absence of bands and lowered the density of the obtained bands in the larvae treated with S. carpocapsae. There were no reports on the activity of EPN toxins on zymographic analysis of enzymes. Although, the changes in band analysis might indicate a change in the enzymes' activities due to the immune-stimulating function of peroxidase in protecting organisms from any stress (Zamocky et al. 2008) or bacteria and parasites infection (Acharya and Ackerman 2014), and the host defence systems of polyphenol oxidases (including, pigmentation and sclerotization) (Malviya et al. 2011).

Conclusions
The above mentioned results revealed that the 5 th instar larvae of A. ipsilon were susceptible to S. carpocapsae, which was more obvious when using 2 Gy gamma-irradiated juveniles. So, it is possible to conclude that gamma-irradiated S. carpocapsae may serve as a safe environmentally safe method for A. ipsilon management under field conditions. Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).
Data availability All data and materials are available if requested.
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