What if an insecticide can stop a crop pest from eating, yet another one leaves the strongest signal of DNA damage? This study compares both effects instead of treating pest control as a single question. The cotton leafworm, Spodoptera littoralis, is a notorious polyphagous pest, causing extensive damage to over eighty-seven plant species from forty families across tropical and subtropical regions.

It threatens worldwide agriculture because of its high reproductive rate and its ability to develop resistance to conventional chemical groups. In Egypt, management of Spodoptera littoralis infestation had previously depended on the traditional application of conventional insecticides.

Unfortunately, heavy and irresponsible application of those chemicals produced ecological issues, including development of resistance, non-target toxicity, and pollution that raises the carbon footprint. The need for a more sustainable and ecologically compatible pest-control method became crucial.

This study investigated the impacts of five biorational insecticides with distinct modes of action on the nutritional physiology of Spodoptera littoralis larvae subjected to a controlled feeding assay. It also evaluated the degree of DNA damage caused by these insecticides, bringing nutritional disruption and genotoxic impacts into the same assessment.

The insecticides ranged from the calcium-channel-targeting chlorantraniliprole to the microbial Bt, allowing the study to examine how these non-conventional insecticides compromise larval fitness. Egg masses provided by the Plant Protection Research Institute and Agricultural Research Center in Giza, Egypt, were used to prepare a laboratory-susceptible colony of Spodoptera littoralis.

After emergence, the larvae were fed fresh castor bean leaves at all times until maturity. The insects were kept in an incubator at a relative humidity of sixty-five plus or minus five percent and a twelve-to-twelve-hour light-dark photoperiod.

A no-choice leaf-disc bioassay was employed to investigate how the selected insecticides influenced feeding activity in Spodoptera littoralis. For each of the five insecticidal products, the LC ten and LC fifty levels were identified.

A concentration gradient was prepared for each insecticide through serial dilution with distilled water. The measurements quantified relative consumption rate, efficiency of conversion of digested food, relative growth rate, approximate digestibility, efficiency of conversion of ingested food, and feeding deterrence index.

All nutritional and feeding parameters were calculated using the formulae developed by Waldbauer. The comet assay was followed using alkaline conditions to analyse DNA single-strand breaks in Spodoptera littoralis larvae exposed to sublethal doses, or LC ten, of the selected insecticides.

The negative control used only water and zero point five percent Triton X one hundred, a non-ionic detergent needed for cell lysis in both control and treated groups. Larval midgut tissue was finely sliced and treated with collagenase to obtain single-cell suspensions, while EDTA inhibited endonuclease activity and DMSO protected DNA against oxidative damage.

Trypan blue staining assessed cell viability, and viable cells that excluded the blue dye were determined using a light microscope. Emamectin benzoate was selected for in silico analysis because it had proved potent against cotton leafworm larvae and was recommended for application by Egypt’s Agricultural Pesticide Committee.

The computational analysis studied possible handling risks by determining toxicity records for tissues that could come into direct contact with the insecticide. The molecular structure and canonical SMILES were obtained from PubChem and submitted to three complementary toxicity-prediction platforms.

The predictions covered AMES mutagenicity, acute oral rat toxicity, chronic oral toxicity, hepatotoxicity, skin sensitisation, Tetrahymena pyriformis toxicity, and minnow toxicity using graph-based molecular signatures in the pkCSM server. Data were checked for normality through the Shapiro-Wilk test and expressed as mean plus or minus standard error.

One-way analysis of variance determined differences among treatments, followed by Tukey’s HSD test as a post hoc multiple-comparison test. Significance was tested at P less than or equal to zero point zero five, and analyses were performed through Minitab software version sixteen point zero, version one.

Feeding deterrence index values were elevated in larvae treated with the insecticides compared with the control, indicating that all tested insecticides strongly deterred feeding. At LC ten, the highest feeding deterrence index occurred with Bacillus thuringiensis at thirty point eight four seven percent and emamectin benzoate at thirty point eight eight nine percent.

Most insecticide treatments significantly reduced relative consumption rate, with indoxacarb recording the lowest value at zero point nine eight nine. At LC fifty, all insecticides raised relative consumption rate except Bacillus thuringiensis, while the tested insecticides generally decreased efficiency of conversion of ingested food.

All tested insecticides significantly increased feeding deterrence index in fifth-instar larvae, indicating a strong effect on deterring larval feeding behavior. Bacillus thuringiensis at LC ten exhibited the highest feeding deterrence at fifty-one point five nine two percent, followed by thiacloprid at LC ten at forty-five point two two percent.

The most considerable decrease in consumption was observed with thiacloprid at LC fifty, at zero point three seven six, while Bacillus thuringiensis at LC ten maintained the highest relative consumption rate at zero point eight four two. Efficiency of conversion of ingested food was predominantly negative, with the most substantial negative impact from thiacloprid at LC fifty at minus sixty point thirty percent, followed by indoxacarb at minus forty-three point ten percent.

Relative growth rate was generally suppressed, and the lowest value was recorded for thiacloprid at LC fifty at minus zero point two one four percent. The statistical rows at the bottom show that LC ten treatments produced significant differences in every measure except AD, with P-values from zero point zero zero one to zero point zero zero four.

At LC fifty, significance remained for ECD, ECI, and RGR, while AD, RCR, and FDI were not significant, as shown by P-values of zero point two zero zero, zero point one nine five, and zero point three seven five. The comet assay used tail length, DNA proportion in the tail, and tail moment to quantify genotoxic damage.

The highest DNA concentration in the tail was detected in cells treated with thiacloprid, whereas the lowest DNA concentration followed Bacillus thuringiensis treatment. Bacillus thuringiensis produced type one or class one damage, corresponding to one to twenty-five percent tail DNA, while all other insecticides scored as type two with twenty-five to forty-five percent tail DNA.

The tail moment showed no overall significant difference between Bacillus thuringiensis treatment and control, but thiacloprid increased tail moment one point five fold, indicating a moderate genotoxic effect. Across all three panels, the letter annotations separate several treatment groups statistically.

In panel A, Coragen and thiacloprid share the letter “a,” while the control and Bacillus thuringiensis bars carry distinct labels; panel C similarly places control and Bacillus thuringiensis together at “d,” with thiacloprid alone marked “a.” ProTox three point zero analysis showed that emamectin benzoate has a mixed toxicological profile.

The insecticide showed immunotoxicity with a probability of zero point ninety-nine and respiratory toxicity with a probability of zero point seventy-eight, identified as the most significant potential safety concerns in the model. Emamectin benzoate was classified as inactive for hepatotoxicity and cytotoxicity, with probabilities of zero point seventy-six and zero point seventy-one, respectively.

The outcomes are computational hazard predictions rather than experimentally validated toxicological findings and should be complemented by genotoxicity, biochemical, and non-target-organism studies. In the probability column, the table spans from zero point fifty-seven for ecotoxicity to zero point ninety-nine for immunotoxicity, a difference of zero point forty-two.

The organ-toxicity rows also show a split classification: two inactive entries and one active entry, while the toxicity-endpoint rows contain one active and two inactive entries. The figure’s left-side inactive cluster connects cyto, eco, neuro, and dili, while the separate active cluster on the right connects immuno and respi.

The red endpoint markers are confined to the active cluster, whereas the inactive cluster contains green markers, with lighter shades indicating probabilities below the threshold. StopTox indicated that the toxicological behaviour of emamectin benzoate differed according to the route of exposure and the endpoint under analysis.

Emamectin benzoate was predicted to be toxic following oral exposure with fifty-two percent confidence and dermal exposure with sixty-one percent confidence. In comparison, the compound was categorised as non-toxic for acute inhalation toxicity with sixty-one percent confidence, and as a non-sensitiser for skin sensitisation with eighty percent certainty.

Because some predictions had moderate confidence, the findings should be viewed as preliminary computational indicators and confirmed using experimentally obtained acute-toxicity and occupational-safety measurements. The fragment-contribution column places molecular structure diagrams beside every endpoint, and the final columns link each prediction to rat, rabbit, or mouse and guinea-pig OECD assay models.

All tested insecticides had a strong feeding-deterrent effect, shown by the substantial increase in the Feeding Deterrence Index of larvae fed on treated plant leaves. Bacillus thuringiensis at LC ten had the highest Feeding Deterrence Index for third- and fifth-instar larvae, demonstrating its strong antifeeding effect.

The high Feeding Deterrence Index values indicate that Bacillus thuringiensis is an effective feeding deterrent against Spodoptera littoralis larvae, supporting its use as a microbial insecticide in pest management. Efficiency of conversion of ingested food and efficiency of conversion of digested food determine how efficiently an insect converts food into body mass.

Many treatments had negative ECI and ECD values, particularly at LC fifty concentrations. Thiacloprid at LC fifty had the lowest ECI value at minus sixty point thirty percent, while the lowest ECD value, minus sixty-one point ten percent, was recorded in fifth-instar larvae.

These findings indicate that the larvae were less efficient in converting food into growth because of the high levels of thiacloprid. The research highlights the importance of analysing several nutritional indices when determining insecticide effectiveness. Bacillus thuringiensis showed a positive profile with moderate sublethal activity against Spodoptera littoralis, whereas thiacloprid and Coragen showed strong deterrent effects alongside substantial disruption to food-conversion efficiency and growth.

The study presents Bacillus thuringiensis as a strong feeding deterrent with moderate sublethal effects, while thiacloprid shows especially strong disruption of food conversion and the greatest DNA damage in the comet assay. Computational toxicity predictions remain preliminary.