Lethal and Sublethal Effects of Insecticides Used in Cotton on Harmonia Axyridis


 The agriculture industry seeks to reconcile control tactics for pest management, necessitating studies of lethal and sublethal effects of insecticides on natural enemies. The effect of six insecticides with diverse modes of action—ethiprole, etofenprox, lambda-cyhalothrin, lambda-cyhalothrin + chlorantraniliprole, pymetrozine, and thiamethoxam + chlorantraniliprole—were evaluated after spraying on eggs, third-instar larvae, pupae, and adults of Harmonia axyridis , a generalist predator in cotton crops. Ethiprole was the only one that had no lethal effect on the predator at any stage of its development; however, it caused sublethal effects when applied to eggs (shortening of the larval phase and lower reproduction). Lambda-cyhalothrin caused high predator mortality after exposure at all stages. Due to the high compatibility between the ladybug H. axyridis and ethiprole, this compound is recommended for use in cotton integrated pest management programs. The other insecticides were harmful in lethal or sublethal tests, and further work in semifield and field conditions is required to confirm if they reduce H. axyridis populations.


Introduction
Cotton crops are attractive to insect pests for many reasons, such as the long period of crop development and its cultivation in large areas (Luo et al., 2014), as well as factors of the plant itself, such as the presence of showy vegetative and reproductive structures, oral exudates, and semiochemicals (Loughrin et al., 1994). These factors stimulate the colonization not only by arthropod pests but also important natural enemies of the pests (Ali et al., 2016). Pests that attack cotton crops have been controlled mainly with chemical insecticides, which represent approximately 20% of the total production cost of cotton (Miranda and Rodrigues, 2016;Wu and Guo, 2005). These compounds usually cause environmental contamination, biological imbalances, resurgence and selection The implementation of integrated pest management (IPM) programs for cotton crops aims to integrate different control tactics to keep pest populations below the economic injury level using the lowest possible pesticide input.
This will result in less impact of the compounds on natural enemies, which are the leading cause of pest natural mortality in agroecosystems (Bueno et al., 2017;Carvalho et al., 2019). In this context, studies on the selectivity of pesticides to natural enemies should be encouraged because they can provide the necessary information on the compatibility of chemical and biological methods within IPM programs of cotton, allowing for greater sustainability of this production sector (Gontijo et al., 2018;Santos et al., 2017). In this context, the lethal and sublethal effects of pesticides applied to this crop should be evaluated to nd ones that best preserve natural enemies (Desneux et al., 2007;Passos et al., 2018).
Predatory insects act as important population regulators of herbivorous arthropods in many agroecosystems (Juen et al., 2012). Coleoptera, belonging to the family Coccinellidae, are polyphagous predators at all stages of their development that feed on various agricultural pests (Farooq et al., 2018; Hodek and Evans, 2012; Oliveira et al., 2004). Among the many species of this family, Harmonia axyridis Pallas (Coleoptera: Coccinellidae) has stood out for its high voracity, as it can feed on approximately 77 species of prey, in addition to its wide distribution in agroecosystems, as it lives on approximately 82 plant species (Honěk, 2012;Koch, 2003). This predator feeds and reduces populations of several aphid species, especially one of the key pests of cotton crops, Aphis gossypii Glover (Hemiptera: Aphididae) (Seko et al., 2014;Iguchi et al., 2012;Xue et al., 2009), as well as other insect pests such as thrips (Li et al., 2020), psyllids (Michaud, 2002), eggs and juvenile stages of Curculionidae (Kalaskar and Evans, 2006;Stuart et al., 2002), and small lepidopteran larvae . Therefore, H. axyridis is arguably an important biological control agent (Riddick, 2017).
Due to the importance of preserving natural enemies in agroecosystems, the present study aimed to evaluate the lethal and sublethal effects of insecticides used on cotton crops towards H. axyridis to search for information that may ensure the compatibility of the chemical and biological methods with this predator in cotton IPM programs.

Materials And Methods
The rearing of H. axyridis and all bioassays were conducted in a climate-controlled room at 24 ± 2°C, 60 ± 10% RH, and a 12-hour photoperiod. The experimental design used for all bioassays was completely randomized.

Insects used in the bioassays
Adult H. axyridis were collected from cotton plants present in the campus of the Federal University of Lavras in Lavras, Minas Gerais, Brazil (latitude: 21° 14′ 45″ south, longitude: 44° 59′ 59″ west). Approximately 200 adults were transported to the laboratory, where they were kept in acrylic cages (60 cm × 30 cm × 30 cm), with side openings lined with voile fabric to allow ventilation and prevent escape. Cabbage leaves (Brassica oleracea L. var. acephala DC.) were kept inside the cages to serve as oviposition substrates. Every 24 hours, the eggs were removed and transferred to Petri dishes 5 cm in diameter, where they remained until the larvae hatched. Larvae were transferred to 20 cm × 10 cm × 10 cm plastic cages and kept until they became pupae, which were placed in Petri dishes to obtain adult insects. Upon emergence, the insects were transferred to an acrylic cage, thus starting a new development cycle.
The larvae and adults of the predator were fed ad libitum every 48 hours with eggs of Ephestia kuehniella (Zeller, 1879) (Lepidoptera: Pyralidae) and Myzus persicae Sulzer (Homoptera: Aphididae) reared in leaves of Nicandra physalodes (Linnaeus) (Solanaceae). Water was provided to the insects via moistened cotton.
Each product was applied via a Potter spray tower, adjusted to a pressure of 15 lb·pol -2 , enabling the application of 1.5 ± 0.5 mg of chemical solution·cm -2 , according to the method established by the International Organization for Biological and Integrated Control of Noxious Animals and Plants (van de Veire et al., 1996).

Effects of insecticides on H. axyridis eggs
For each treatment, 70 eggs (≤24 hours old) of the predator, obtained from laboratory rearing, were distributed at a ratio of 14 per 10-cm-diameter Petri dish and were treated by spraying in a Potter spray tower. The Petri dishes were then sealed with polyvinyl chloride (PVC) lm with small holes to allow gas exchange.
The seven treatments evaluated were done in ve replicates, each consisting of a Petri dish with 14 eggs of the predator. The evaluations began 24 hours after insecticide spraying. We recorded the duration of the embryonic period and the viability of the treated eggs. To avoid cannibalism, 48 hours after hatching, the surviving larvae were put in individual Petri dishes of 5-cm diameter that were sealed with PVC lm. They were fed ad libitum with E. kuehniella eggs every 48 hours, and water was provided through moistened cotton. The following parameters were analyzed: durations of the larval and pupal stages, pupal viability, numbers of males and females for determining the sex ratio, and longevity of adult females.
To evaluate the effects of the products on reproduction, at least ve pairs of surviving adults were separated per treatment (Hassan 1977), which were put individually in Petri dishes of 10-cm diameter, fed, and kept under the same conditions mentioned above. After the preoviposition period, to avoid predation of the eggs, the males were removed and weighed, and the total number of eggs (total fecundity) laid during the rst ve consecutive ovipositions were recorded. To evaluate viability, at least 40 eggs from the third and fourth egg lays in each replicate were put individually in Petri dishes. At the end of the evaluations of the number of eggs laid, the adult females were weighed.

Effect of insecticides on H. axyridis larvae
Forty third-instar larvae per treatment were treated by spraying the insecticides in a Potter spray tower and were then put individually in PVC-sealed Petri dishes 5 cm in diameter. Survival was evaluated daily up to 96 hours after spraying the insecticides, and the duration of the larval and pupal phases and pupal viability were also evaluated.
To evaluate the effects on the reproduction of adults originating from treated larvae, at least ve surviving couples were separated per treatment (one couple per replicate), which were individualized into 10-cm-diameter Petri dishes. After the preoviposition period, the total number of eggs laid during the rst 5 days was recorded to calculate the fecundity of the females, and then the adults were weighed. To assess egg viability, at least 40 eggs from the third and fourth ovipositions were individualized in Petri dishes.

Effects of insecticides on H. axyridis pupae
A total of 40 pupae aged up to 24 hours were subjected to each treatment. They were distributed at a total of ve per Petri dish and treated in a Potter spray tower as above. Next, each pupa was put into its own Petri dish 5 cm in diameter, and after the emergence of the adults, they were kept and fed as described above.
The seven treatments had ve replicates, and each plot consisted of eight pupae. The duration of the pupal phase, pupal viability, and morphological deformities in the wings of adult insects were evaluated. To observe the possible effects of insecticides on H. axyridis reproduction, at least ve pairs were formed, which were allocated at one pair per 10-cm Petri dish to perform the evaluations. The biological characteristics evaluated were the same as those described in the third-instar bioassay.

Effect of insecticides on H. axyridis adults
For each treatment, 40 adults up to 48-hours old were distributed at ve per Petri dish and treated with the products via Potter tower spraying. Soon after the application of the insecticides, the adults were transferred to new Petri dishes 10 cm in diameter, where they were kept and fed as above. Insect survival was evaluated daily up to 96 hours after application of the products. To evaluate the effects of the compounds on the reproduction of the surviving adults, at least ve couples were separated per treatment (each couple represented one replicate) into 10-cm Petri dishes, and the evaluations were performed as above.

Data analysis
Data on the number and viability of eggs and pupae, duration of the larval period, larval survival, duration of the pupal period, adult weight, morphological deformities in the wings of the insects, and duration of the embryonic period were checked for homoscedasticity and normality to test the assumptions of analysis of variance (ANOVA). Next, they were subjected to one-way ANOVA with the Scott-Knott test (p < 0.05) (R Development Core Team, 2016). When the data did not have a normal distribution, they were tted to generalized linear models (GLMs) using other distributions, such as Poisson and binomial, and the means were subjected to pairwise comparison (R Development Core Team, 2016). Sex ratio data were analyzed by the chi-squared test at the p < 0.05 level (Prop test, R Development Core Team, 2016).
The survival data of larvae and adults were subjected to survival analysis following the Weibull distribution model ('Survival', R Development Core Team, 2016). This procedure estimated survival curves using least squares estimators without censoring, generated from the proportion of surviving adults or larvae. To draw the graph, the following equation was used: f(x) = exp(-µ -α t α ), where α in the software R is given by 1/Scale, where Scale is the value returned by the command "summary", and µ is the 50%-lethal time (mean time for achieving the death of half of the treated population) in each treatment.

Effects of insecticide application on H. axyridis eggs
When the insecticides were applied to H. axyridis eggs, none of them shortened the embryonic period, which averaged 2.4 to 2.8 days (p = 0.9201; df = 6; F = 0.3216). Lambda-cyhalothrin + chlorantraniliprole, etofenprox, and lambda-cyhalothrin reduced egg viability, differently from the other insecticides ( Figures 1A and 2).
Despite the high percentage of hatching in the treatments with thiamethoxam + chlorantraniliprole, pymetrozine, and etofenprox, the survival of the larvae after 48 hours was less than 10%, making it impossible to evaluate other biological parameters. All larvae that hatched from eggs treated with lambda-cyhalothrin + chlorantraniliprole or lambda-cyhalothrin died within 48 hours after hatching. Ethiprole did not reduce larval survival, allowing evaluations of other biological parameters ( Figures 1A and 2).
The larval stage was shortened in the ethiprole treatment, but this insecticide did not reduce pupal viability, adult weight (females and males), preoviposition period, total fecundity, or sex ratio. Nevertheless, ethiprole decreased egg viability of treated adults ( Table 1).
For the three insecticides that caused the highest mortality, evaluations of sublethal effects were impossible. Of the evaluable insecticides, only etofenprox prolonged the larval stage ( Table 2). The duration of the pupal stage, pupal survival, adult female weight, duration of the preoviposition period, and total fecundity were not reduced by any insecticide. The adult male insects that grew from larvae treated with pymetrozine or etofenprox had reduced body weight, whereas ethiprole had no such effect. Egg viability was reduced only by etofenprox ( Table 2).

Effects of insecticide application on H. axyridis pupae
The pupal period was prolonged in the treatments with lambda-cyhalothrin + chlorantraniliprole, etofenprox, and lambda-cyhalothrin. The survival of insecticide-treated pupae was not reduced by thiamethoxam + chlorantraniliprole, pymetrozine, ethiprole, or etofenprox ( Figure 1C). Lambda-cyhalothrin + chlorantraniliprole and lambda-cyhalothrin reduced pupal survival, in addition to causing morphological deformities in the elytra and di culting exuvia remotion ( Figure 1C and Figure 3). Two days after emergence, the insects of these two groups began to die, and after 5 days, fewer than 10% of them were alive, which differed from the other groups ( Figure 4).
Due to the high mortality caused by lambda-cyhalothrin + chlorantraniliprole and lambda-cyhalothrin, we could not evaluate their effects on reproduction. The thiamethoxam + chlorantraniliprole treatment was the only one that prolonged the preoviposition period, while the other treatments did not affect this biological parameter. Fecundity and egg viability were reduced by thiamethoxam + chlorantraniliprole and pymetrozine, whereas ethiprole had no effect ( Table 3).

Effects of the application of insecticides on H. axyridis adults
Lambda-cyhalothrin + chlorantraniliprole and lambda-cyhalothrin were the only insecticides toxic to H. axyridis adults ( Figure 1D

Discussion
In the present study, the insecticides affected the biological parameters of H. axyridis at varying degrees. Among the life stages of the predator, higher levels of toxicity were observed when eggs or third-instar larvae were exposed to insecticides, showing that these developmental stages are more sensitive to these insecticides than pupae or adults. The greater tolerance of adults to insecticides may be related to the higher sclerotization of their tegument (Nation, 2015), as greater rigidity and waxiness may reduce the penetration capacity of chemical compounds. Other important factors are the changes in the detoxi cation enzyme activity and sensitivity of the action site that occur over the insect life cycle. In the study by Cho et al. (2002), the concentration of an organophosphate (phosphamidon) required for acetylcholinesterase inhibition was 183.5 times higher in H. axyridis adults than in larvae, in addition to a 6.2-fold increase in the enzymatic activity of glutathione Stransferase. In addition, the weight of insects increased throughout development (egg to adult), reducing the proportion of insecticide by insect weight. This variation in toxicity between life stages has also been observed in H. axyridis in response to other insecticides and in other coccinellid species, such as Eriopis conna (German) and Adalia bipunctata (Linnaeus) (Coleoptera: Coccinellidae) (Fogel et al., 2013;Garzón et al., 2015), where the larvae of these predators were more susceptible to insecticides than adults.
Ovicidal action occurs when the insecticide does not allow the larvae to hatch, despite the development of the embryo, which can be observed as a change in egg color from yellow to black (Koch, 2003). The physical and chemical structures of the egg chorion promote protection against the dissection and entry of hydrophilic substances during embryo development (Nation, 2015). The ability of insecticides to penetrate the chorion depends on their octanol-water partition coe cient (log K o/w ), and those with high log K o/w values more easily cross the chorion and translocate to the site of action (Hoffmann et al., 2008). In the present study, insecticides with higher log K o/w , such as lambda-cyhalothrin (log K o/w = 7.0) and etofenprox (log K o/w = 6.9), had an ovicidal effect. The insecticides with lower lipophilicity, pymetrozine (log K o/w = -0.18), thiamethoxan + chlorantraniliprole (log K o/w = -0.13 and 2.8), and ethiprole (log K o/w = 2.9), might have been blocked by the wax layer of the chorion and were therefore innocuous to the egg.
We assumed that lambda-cyhalothrin caused the ovicidal effect of the lambda-cyhalothrin + chlorantraniliprole mixture since this compound has high lipophilicity and, when evaluated alone, also caused a low hatching rate. Chlorantraniliprole belongs to the chemical group of anthranilic diamides, and due to its lower lipophilicity (log K o/w of 2.8), it must have had di culty penetrating the chorion. Yoriatti et al. (2009) found a low ovicidal effect of chlorantraniliprole when eggs of Lobesia botrana (Denis & Schiffermüller) (Lepidoptera: Tortricidae) were directly exposed to its residues. The habit that coccinellid larvae have of feeding on and remaining on the chorion up to 24 hours after hatching (Nedvěd and Honěk, 2012) may have favored their contamination and consequently caused their death soon after they hatched. Similar results are reported for H. axyridis, in which thiamethoxam did not reduce egg viability but caused high mortality of rst-instar larvae, and in which etofenprox reduced egg viability and caused high mortality among newly hatched larvae (Youn et al., 2003).
Unlike the other insecticides, ethiprole did not cause mortality of newly hatched larvae but shortened the larval period, and the adults generated laid eggs with reduced viability. Adults with inadequate nutritional status have reduced egg quality and viability (dos Santos Rodrigues et al., 2013;Shapiro and Shirk, 2010). The shortening of the larval stage may have led to malnourished adults, which resulted in reduced egg viability.
Thiamethoxan + chlorantraniliprole, pymetrozine, and etofenprox caused lower mortality of third-instar H. axyridis larvae than rst-instar larvae that hatched from treated eggs. Often, insects in their rst instars are more susceptible to the effects of insecticides as a result of their lower enzymatic activity (Cho et al., 2002).
Like the chorion, the tegument of insects also has a protective wax layer that provides impermeability (Nation, 2015). Thus, the speci c physicochemical characteristics of each insecticide may provide greater or lesser penetration through the tegument. Lambda-cyhalothrin, lambda-cyhalothrin + chlorantraniliprole, and etofenprox reduced the survival of H. axyridis larvae, probably due to their high log K o/w values (> 6.9), which may have contributed to their greater penetration of the tegument and thus greater likelihood of reaching its site of action. Although etofenprox is also a pyrethroid, the results of our study show that this active ingredient is less harmful to H. axyridis than lambda-cyhalothrin since most of the larvae recovered from the knockdown effect and the insecticide did not cause mortality in pupae or adults of this predator. However, it lengthened the larval stage and reduced the total fecundity and viability of the eggs of the adults. It is noteworthy that the larvae and adults that suffered a knockdown effect of etofenprox recovered within 48 hours.
The different results found for the insecticides might be related to the different metabolism and/or excretion routes due to variations in the chemical structures of the insecticides, even in the case of compounds belonging to the same chemical group (Nauen et al., 2015). The main enzymes involved in pyrethroid tolerance are cytochrome P450 and esterases, as they catalyze multiple oxidative reactions due to increased gene expression of this family of enzymes (Scott and Wen, 2001). In Propylaea japonica (Thunberg) (Coleoptera: Coccinellidae), the increase in the activity of the enzymes glutathione S-transferase, carboxylesterase, and cytochrome P450 monooxygenase is reported to be involved in greater tolerance to insecticides (Wu et al., 2018).
The energy expenditure needed to detoxify etofenprox may have been responsible for the prolongation of the larval phase of the predator and negative effects on the nutritional status of the emerged adults, which ultimately culminated in decreased viability and fecundity of eggs. These results corroborate those of Galvan et al. (2005), who also observed a longer young stage of H. axyridis when the insects were treated by spraying the insecticide indoxacarb. They assumed that such an effect this was resulted from metabolism and/or energy expenditure required for the insecticide excretion. Santos et al. (2016) found that E. connexa ladybug recovered from the knockdown effect of pyrethroid insecticides within up to 72 hours.
The sublethal effects (reduced weight of males, reduced fertility, and reduced egg viability) caused by pymetrozine when sprayed on larvae, pupae, and adults, are related to its mode of action. This insecticide blocks the salivary gland of insects, causing the interruption of food intake mainly in sucking insects (Sechser et al., 2002). Cunha et al. (2015) found that Coleoptera were also sensitive to pymetrozine, with histological changes in the intestine. Thus, the reduced weight of adult males may have been due to these effects of the insecticide, leading to reduced food consumption of H. axyridis. Although only the weight of males changed, this may explain the reduction in egg viability because males with higher weight may supply a more sperm, oviposition stimulants, and other nutrients during copulation than smaller males (Omkar and Afaq, 2013;Singh et al., 2019). According to Almasi et al. (2013), pymetrozine caused close to 40% mortality of third-instar larvae of the ladybug Hippodamia variegate (Goeze) (Coleoptera: Coccinellidae). On the other hand, when applied to adults of Coccinella septempunctata Linnaeus and H. variegata (Coleoptera: Coccinellidae), pymetrozine was shown to be harmless (Skouras et al., 2019).
Overall, the pupal stage was the most tolerant to the insecticides in this study since only one insecticide (lambdacyhalothrin) reduced pupa survival. This compound also led to deformities in adult elytra. In addition, when applied in pupae, thiamethoxam, pymetrozine, ethiprole and etofenprox affected the pupal period, adult fecundity and egg viability. These negative effects may be related to the interference of insecticides on the nervous system of insects, which is responsible for regulating the synthesis of neurohormones and different enzymatic processes, which affect the production of hormones related to development and metamorphosis. Li et al. (2019) found that changes in the production of the enzyme α-glucosidase-trehalose in H. axyridis impair the chitin synthesis pathway, hindering insect development and causing wing deformities. Corroborating the results obtained in the present work, other studies have demonstrated that H. axyridis pupae exposed to thiamethoxam or etofenprox had reduced survival (Youn et al., 2003). However, in E. connexa pupae, the application of cypermethrin signi cantly reduced their survival and led to the formation of adults with morphological deformities in the wings (Fogel et al., 2016). The high mortality after the emergence of the deformed insects in our study probably occurred due to dehydration and starvation since they had di culty moving and feeding.
In the adult stage, only lambda-cyhalothrin and lambda-cyhalothrin + chlorantraniliprole were toxic to H. axyridis. Possibly, the high mortality caused by the lambda-cyhalothrin + chlorantraniliprole mixture is due only to the active ingredient lambda-cyhalothrin, since the numbers of dead insects with and without chlorantraniliprole were similar. In addition, studies have shown the selectivity of chlorantraniliprole for H. axyridis  and other natural enemies of the Coccinellidae family, such as C. septempunctata (He et al., 2019;Jiang et al., 2020). Lambda-cyhalothrin has a broad spectrum of action and low selectivity for natural enemies of the Coccinellidae family. When larvae of H. axyridis and A. bipunctata were exposed to a surface treated with lambda-cyhalothrin, the mortality was higher than 80% (Benelli et al., 2015).
In short, the early stages of development of H. axyridis, such as eggs and larvae, were the most susceptible to insecticides, followed by the pupal and adult stages. The present results can help IPM programs achieve compatibility between the chemical and biological methods, through the use of predator-friendly insecticides. In this context, the insecticide that showed the greatest compatibility with the predator was ethiprole, which may be recommended in IPM programs of cotton. The other insecticides were toxic and should be evaluated under semi eld and eld conditions to con rm their deleterious effects on H. axyridis. Means (± SE) followed by the same letter in a column do not differ by the ttest (p < 0.05). *At least ve couples were evaluated per treatment.  Means (± SEM) followed by the same letter in a column do not differ. One-way ANOVA (p < 0.05) or 1 GLM (p < 0.05). * Time recorded after treatment application. At least ve couples were used per treatment. ** Not enough insects were obtained to evaluate the biological parameters. Figure 1 a) Hatching and survival (mean ± SEM) of larvae from eggs of Harmonia axyridis treated with insecticides. Different capital letters indicate differences in black bars (quasi-binomial GLM, pairwise comparison, p < 0.05), and different lowercase letters indicate differences between gray bars (one-way ANOVA, p < 0.05). b) Survival (mean ± SEM) of third-instar larvae of H. axyridis 96 hours after treatment with insecticides. Different letters indicate differences between treatments (one-way ANOVA, p < 0.05). c) Survival (mean ± SEM) of pupae of H. axyridis after treatment with insecticides. Different letters indicate differences between treatments (one-way ANOVA, p < 0.05). d) Survival (mean ± SEM) of pupae of H. axyridis after treatment with insecticides. Different letters indicate differences between treatments (quasi-binomial GLM, pairwise comparison, p < 0.05).

Figure 2
a) Live Harmonia axyridis larvae newly hatched from eggs treated with water or ethiprole. b) Dead larvae a few hours after hatching from eggs treated with thiamethoxam + chlorantraniliprole, pymetrozine, or etofenprox. 2c) Dark eggs, characterizing embryonic development, but no larvae hatched due to the effect of lambda-cyhalothrin + chlorantraniliprole or lambda-cyhalothrin. Photos taken under a stereoscopic microscope (35×).

Figure 3
a) Pupa and adult Harmonia axyridis treated with water (control): no morphological deformity in the elytra. Adults with elytral deformity were unable to remove the exuvia in the lambda-cyhalothrin + chlorantraniliprole group (b) and lambda-cyhalothrin group (c). Photos taken under a stereoscopic microscope (25×). Scale bars: 1 cm.

Figure 4
Survival curves (adjusted Weibull model) of adult Harmonia axyridis that grew from pupae treated with insecticides in a Potter spray tower. The curve of group 1 applies to the control (water), thiamethoxam + chlorantraniliprole, pymetrozine, ethiprole, and etofenprox treatments, with a half-lethal time of 17.46 days, while the curve of group 2 represents the lambda-cyhalothrin + chlorantraniliprole and lambda-cyhalothrin treatments, with a half-lethal time of 4.43 days.