Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae) is a serious insect pest that damages many vital crops across the world. During their development, A. ipsilon larvae may eat up to 400 cm2 of vegetation. Chemical pesticides have been commonly used to prevent A. ipsilon from destroying crops.
Image Credit: Andrii Medvediuk/Shutterstock.com
Nanotechnology may be able to provide alternative technologies to alleviate concerns regarding the negative environmental consequences of using chemical pesticides. Increased exposure and toxicity of these chemicals can affect non-target organisms and ecosystems. Nanoparticle technology might evolve in two ways: (a) as a single crop protection system, or (b) as pesticide carriers.
Insecticide research focuses on the effects of chemically active substances on insect growth and enzyme activities, both deadly and sublethal. Insecticides disrupt an insect’s functional balance (oxidative stress), by producing more reactive oxygen species (ROS) while impairing their scavenging mechanisms.
Insects have significant antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), and glutathione (L-glutamyl-L-cysteinylglycine, GSH). Superoxide dismutase (SOD) is an antioxidant enzyme that breaks down superoxide into oxygen and hydrogen peroxide. In all aerobic organisms, CAT is primarily an H2O2-scavenging enzyme that eliminates H2O2 produced by developmental or environmental stimuli into water and oxygen.
POX can oxidize a wide range of molecules with either H2O2 or O2, with H2O2 being used to oxidize phenolic compounds. GSH is an electron donor (cofactor) for antioxidant enzymes such as glutathione peroxidases and glutathione S-transferases. The inter-simple sequence repeats (ISSR) are a valuable marker for detecting genetic variation and distinguishing closely related people.
In new research published in the journal PLoS ONE, researchers investigated how A. ipsilon responded to nano-forms of two novel insecticides, chlorantraniliprole and thiocyclam. The study also investigated the effects of lethal and sublethal doses of both insecticides and their nano-forms on A. ipsilon development, reproduction, and oxidative stress enzyme activities including SOD, CAT, lipid peroxidase, and GR.
In the laboratory, Agrotis ipsilon was maintained for 12 generations without pesticides in a rearing chamber. Researchers evaluated chlorantraniliprole and thiocyclam.
The finished nano-suspension was ready for TEM analysis. The toxicity of chlorantraniliprole, thiocyclam, and their nano-forms on second instar larvae was determined using a modified leaf dipping approach. On the second instar larvae, lethal dosages of chlorantraniliprole and thiocyclam were used to annihilate 15 and 50% of the larvae exposed to each form.
The total protein content of all samples was determined spectrophotometrically. Following the manufacturer’s instructions, DNA was extracted from both untreated and treated second instar larvae using a G-spinTM total DNA extraction kit.
Top-down molecular chemical techniques were used to create nano-suspensions of chlorine in Figure 1 and Sulfur in Figure 2.
Figure 1. Nano-chlorine. Image Credit: Awad, et al., 2022
Figure 2. Nano-sulfur. Image Credit: Awad, et al., 2022
Table 1 shows the irregular form of the particles, with diameters of 4.05 nm for sulfur and 3.99 nm for chlorine, as well as 98.5% purity for each element.
Table 1. Particle size of nano-chlorine, and nano-sulfur. Source: Awad, et al., 2022
Table 2 displays the toxicity of chlorantraniliprole, thiocyclam, and their nano-forms on second instar larvae.
Table 2. Lethal and sublethal activity of chlorantraniliprole, thiocyclam, and their nano-form in the 2nd instar larvae of Agrotis ipsilon. Source: Awad, et al., 2022
Table 3 indicates the latent impact of thiocyclam, chlorantraniliprole, and their nano-forms on A. ipsilon development owing to LC15 and LC50 exposure.
Table 3. Effects of chlorantraniliprole, thiocyclam, and their nano-form in the developmental stages of A. ipsilon. Source: Awad, et al., 2022
Compared to the control, chlorantraniliprole, thiocyclam, and their nano-forms drastically reduced hatchability % at LC15 and LC50 in Table 4.
Table 4. Mean fecundity and hatchability % (±SE) of A. ipsilon females after treating the 2nd instar larvae with LC15 and LC50 values of chlorantraniliprole, thiocyclam, and their nano-form. Source: Awad, et al., 2022
Following treatment with the LC50 of nano-thiocyclam (43.0 U/g of protein) and thiocyclam (40.09 U/g of protein), SOD activity increased significantly after exposure to both insecticides and their nano-forms as shown in Table 5.
Table 5. Mean (±SE) of oxidative stress enzymes (SOD, CAT, glutathione reductase, and lipid peroxidase) activities of A. ipsilon after exposure of 2nd instar larvae to LC15 and LC50 values of chlorantraniliprole, thiocyclam, and their nano-forms. Source: Awad, et al., 2022
The silhouette analysis plots were created using the Euclidean distance metric to evaluate the cluster quality of the treatments on the basis of reproductive activity, enzymes, and developmental parameters using cluster distance tests within and between each cluster are depicted in Figure 3.
Figure 3. Plot of Silhouette analysis values for clustering of reproductive activity, enzymes, and developmental variables. On the y-axis each cluster are ordered by decreasing silhouette value. The silhouette value can range between −1 and 1. Image Credit: Awad, et al., 2022
The nano-thiocyclam LC15 was classified as less comparable to the control in the second cluster are seen in Figure 4.
Figure 4. Two-dimensional heatmap visualization shows the interaction between the treatments and (A) the eight developmental parameters (B) the two reproductive activity parameters (C) the four enzymes’ parameters. Image Credit: Awad, et al., 2022
Figure 5 displays the effects of multidimensional preference analysis, which highlighted the interrelationships of all treatments, parameters, and classes.
Figure 5. Multidimensional preference analysis plot summarizing the interrelationships amongst treatments, parameters, and classes. Image Credit: Awad, et al., 2022
15ISSR primers were used to amplify the DNA of the untreated second instar larvae and other treatments (Figure 6). With an average of 16.8 bands per primer, the 15 ISSR primers produced a total of 252 scoreable amplicons are displayed in Table 6.
Figure 6. A representative agarose gel where PCR products of the 15 ISSR primers for the nine treatments. Image Credit: Awad, et al., 2022
Table 6. Primer names, number of total bands, polymorphic bands, percentage of polymorphism, and markers efficiency parameters of ISSR primers. Source: Awad, et al., 2022
Table 7 shows the lowest GS, which was discovered to lie between the LC50 of nano-chlorantraniliprole and nano-thiocyclam.
Table 7. Genetic similarities between the nine treatments based on Jaccard’s similarity coefficient based on ISSR primers data. Source: Awad, et al., 2022
For the nine treatments, a dendrogram was created using UPGMA cluster analysis of the ISSR data is illustrated in Figure 7.
Figure 7. UPGMA cluster analysis based on Jaccard’s similarity coefficient of ISSR analysis of the nine treatments: C; Control, C15; chlorntraniliprole LC15, C50; chlorntraniliprole LC50, Cn15; nano-chlorntraniliprole LC15, Cn50; nano-chlorntraniliprole LC50, T15; thiocyclam LC15, T50; thiocyclam LC50, Tn15; nano-thiocyclam LC15 and Tn50; nano-thiocyclam LC50. Image Credit: Awad, et al., 2022
Figure 8 shows the PCA findings that showed the LC15 of nano-chlorantraniliprole and the LC50 of thiocyclam were the most comparable to the control.
Figure 8. A representative agarose gel where PCR products of the 15 ISSR primers for the nine treatments. Image Credit: Awad, et al., 2022
The ISSR-PCR approach was utilized as an efficient marker to evaluate the degrees of genetic mutagenicity in second instar larvae exposed to several insecticidal treatments (chlorantraniliprole, thiocyclam, and their nano-forms) to the untreated control. Compared to the control, the LC15 of nano-chlorantraniliprole treatment had the least mutagenic insecticidal effects on insect DNA.
The LC50 of nano-chlorantraniliprole had the highest mutagenic impact, followed by the LC50 of nano-thiocyclam. The PCA analysis of ISSR data yielded a similar outcome to the dendrogram topology. Furthermore, the mutagenic effects of nano-chlorantraniliprole LC15 and thiocyclam LC50 were shown to be nearly identical to the control.
The polymorphic variances in ISSR patterns are thought to be due to alterations in primer binding sites or DNA architecture, as well as DNA damage caused by pesticide treatment. Furthermore, it might be caused by DNA replication blockage, the existence of a significant number of chromosomal lesions, such as major rearrangements (e.g., deletion, inversion, or translocation), or unrepaired DNA damage caused by direct insecticide exposure.
The ISSR investigations proved their capacity to identify DNA damage and alterations induced by the pesticides tested.
To summarize, insecticide monomerization was created to increase the biological activity of traditional insecticides. The findings are a potential step forward in the development of safe and effective nano-insecticides. More research is needed to understand their environmental impact. This study is the first of its kind to look at the genome-wide DNA mutability, biochemical impacts, and toxicity levels of thiocyclam, chlorantraniliprole, and their nano-forms for A. ipsilon control.
Awad, M., Ibrahim, E. D. S., Osman, E. I., Elmenofy, W. H., Mahmoud, A. W. M., Atia, M. A., Moustafa, M. A. (2022). Nano-insecticides against the black cutworm Agrotis ipsilon (Lepidoptera: Noctuidae): Toxicity, development, enzyme activity, and DNA mutagenicity. PloS one, 17(2), p. e0254285.
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