Effect of Insect Growth Regulator Insecticides Novaluron, Teflubenzuron and Lufenuron on the Morphology and Physiology of Euschistus heros

The Neotropical brown stink bug, Euschistus heros (F.) (Heteroptera: Pentatomidae), is an important pest that damages soybean, especially in the central-west region of Brazil. The effect of insect growth regulator insecticides on fourth-instar nymphs and adults of the neotropical brown stink bug was evaluated under laboratory and greenhouse conditions, respectively. In the laboratory, the insecticides (doses in g a.i. ha -1 ) novaluron (20.0 and 40.0), teflubenzuron (26.2 and 52.5), and lufenuron (20.0 and 40.0), in addition to the control (water), were tested on fourth-instar nymphs of E. heros and their development were followed to adulthood. Mortality, number of adults with and without deformities, and fecundity were determined. In a greenhouse, the effects of the same insecticides on the adult of the stink bug were evaluated by determining the fecundity and viability of the eggs laid. The fourth-instar nymphs of E. heros , when exposed to either dose of the tested insecticides presented mortality and insects with deformations, as well as reduced fecundity of females that reached adulthood. In the trial with adult E. heros , all tested insecticides reduced stink bug fecundity. In the same way, the viability of the produced eggs was reduced in all the chemical treatments, except for novaluron at the lowest dose tested, in which egg viability did not differ from the control treatment. Based on the obtained results, one can infer that the growth-regulator insecticides evaluated, although usually more suitable for the control of caterpillars, can interfere negatively in the development and reproduction of the neotropical brown stink bug, thus constituting a complementary alternative for the management of this pest.


Introduction
The crops Glycine max (L.) soybean and Zea mays L. corn occupy positions of economic prominence in the countries that produce them, and their grains are marketed and consumed worldwide by both humans and animals (CONAB, 2023). Brazil assumed the title of largest producer and exporter of soybean in the world, with production in the 2022/2023 harvest of 153,633.0 million tons (CONAB, 2023). However, numerous phytosanitary problems can negatively affect the productivity of these crops in Brazil, especially diseases and insect pests, which can cause severe damage throughout the crop development cycle (Possebom, Lucini & Panizzi, 2020).
The control of Neotropical stink bugs in soybean crops has mostly been carried out through spraying of chemical insecticides belonging to the groups of neonicotinoids, pyrethroids, carbamates and organophosphates (Castellanos et al., 2019). In corn, the same groups of insecticides are used in spraying, in addition to seed treatment, especially with neonicotinoids (Crosariol Netto et al., 2015). However, continuous exposure of stink bugs to insecticides promotes the development of insect populations resistant to these products (Sosa-Gómez et al., 2001;Sosa-Gómez & Silva, 2010;Guedes, 2017;Tuelher et al., 2018;Somavilla et al., 2019). Thus, alternatives to control phytophagous stink bugs in crops that are compatible with the principles of Integrated Pest Management (IPM) are both economically and environmentally desirable (Liu et al., 2002).
Physiological insecticides, also known as Insect Growth Regulators (IGRs), belong especially to the benzoylurea group and are part of compounds that act on the immature stage of lepidopterans, generally preventing the target species from reaching the adult stage (Sun et al., 2015;Meng et al., 2020). This group of insecticides is normally characterized as selective products for nontarget organisms and have low toxicity for mammals (Khorshidi et al., 2019). Some IGR insecticides can cause negative effects on the reproduction of insects in the orders Coleoptera (Ávila & Nakano 1999;Nakano et al., 2018), Hemiptera (Furiatti et al., 2009;Zantedeschi et al., 2017), Orthoptera (Ghazawy, 2012), and Diptera (Djeghader et al., 2014). Thus, the morphological and physiological effects of IGRs on different groups of pests need to be evaluated to better understand the action of these products on the development of insects.
Therefore, the effects of the growth-regulating insecticides novaluron, teflubenzuron, and lufenuron on fourth-instar nymphs and adults of E. heros were evaluated, considering aspects of development and reproduction. A deeper understanding of the effects of IGRs on the development of E. heros can be justified by the fact that these insecticides can cause indirect effects on this insect, due to their occurrence in the same soybean structure as the lepidopterans that are direct targets of IGRs.
Two experiments were conducted with E. heros-one with fourth-instar nymphs under laboratory conditions and the other with adults in a greenhouse. The minimum and maximum dose of the IGRs novaluron, lufenuron, and teflubenzuron recommended for the control of caterpillars in the soybean crop was used as treatments in both trials, in addition to an untreated control (Table 1).

IGRs Effect on Euschistus heros Nymphs
The experiment was conducted in the laboratory under controlled conditions of temperature: 25±1 °C, relative humidity: 70±10%, and photophase: 14 hours. Fourth-instar nymphs of E. heros up to 24 h old, from the laboratory rearing, were used.
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Statisti
The data homosceda F test, the  Vol. 15,No. 7; all the experim in 5-L plastic fter soybean p when the soy eatments previo up to 24 hours lants as well a (Figure 2 Figure 3A), little locomotion, and difficulty returning to their natural position after some disturbance. Similarly, other nymphs had marked deformation of the head and lateral extensions of the pronotum ( Figure 3B-3C) or kept their exuvia retained in the posterior region of the body ( Figure 3D). In addition, some nymphs presented a liquid blister on their dorsal part, and some adults from the treated nymphs presented deformations or difficulties ridding themselves of their old cuticle ( Figures 3E-3F).

Mortality
The mean number of normal adult E. heros from the nymphs exposed to IGRs were lower in all treatments than the control, although without significant difference between the treatments ( Table 2). As a result, control of 83.3% to 100% of adult stink bugs were achieved in the treatments with IGRs ( Table 2). The E. heros females exposed as nymphs to IGRs laid a relatively very low average number of eggs in the two treatments with teflubenzuron compared to the control treatment. Females in the treatments with the other insecticides presented no fecundity (Figure 4).  The E. heros couples maintained in contact with soybean plants sprayed with the different IGRs produced a lower number of ovipositions than the control treatment, with the exception of the lufenuron treatment (20.0), in which the oviposition was similar to the unsprayed plants (Table 3). However, the average number of eggs obtained from couples exposed to IGRs was lower in all treatments with insecticides than the control, without however differing between them (Table 3). Means followed by the same letter do not differ statistically by Tukey's test (p < 0.05).
The viability values of eggs obtained from E. heros couples that had contact with plants sprayed with IGRs were also lower in most chemical treatments when compared to the control ( Figure 5). The exception was the treatment with the lowest dose of novaluron in which the viability of the eggs was similar to the control ( Figure  5). Eggs from couples exposed to IGRs generally contained empty interiors or mortatlity embryos, which reflected negatively on their viability.

Discussion
The results obtained with the IGRs tested on E. heros generally indicated that they caused deleterious effects on the immature phase of this species, affecting the normal process of ecdysis and causing deformations, and mortality of E. heros nymphs.
Studying the effect of different concentrations of lufenuron (250, 300, 350, and 400 mg.ml -1 ) on E. heros, Turchen et al. (2016) observed that this IGR at concentrations of 250 to 400 mg.ml -1 also caused the mortality of the neotropical brown stink bug nymphs as well as reduced the number of nymphs that completed the ecdysis process until adulthood. These authors also verified that lufenuron did not cause adult mortality or interfere with the fecundity and viability of stink bug eggs. These results partially corroborate to those obtained in this work, as the tested doses of lufenuron also caused mortality of nymphs of the stink bug.
Insecticides belonging to the chemical group of benzoylureas are considered inhibitors of chitin synthesis and act on the ecdysis process during the immature phase of lepidopterans, when they prevent the secretion of a new cuticle and exocuticle release in the caterpillars, thus causing their mortality (Aliabadi et al., 2016). Similar results were observed in this work for the nymph ecdysis process, mortality, deformations, changes in the number of ovipositions, egg viability, in E. heros nymphs, although these insects are in a different order (Matsumoto et al., 2021;Cremonez et al., 2019). In a study with the insecticides lufenuron (0.187 g a.i. ha -1 ) and novaluron (0.249 g a.i. ha -1 ) on the immature phase of Anticarsia gemmatalis Hubner 1818 (Lepidoptera: Eribidae), Loeck et al. (2007) found deleterious effects in the prepupal stage and deformations in the wings of adults, as well as a reduction in the females fecundity. Similar results were observed in this work for the fourth-instar nymphs of the Neotropical brown stink bug, when submitted to lufenuron and novaluron, which caused deformation and mortality of E. heros nymphs, in addition to the negative effects on the fecundity and viability of eggs produced by this species. For nymphs of E. heros, Lufenuron and diflubenzuron caused malformations in the mouthparts, consequently inhibiting feeding and due to this, nutritional deficiency directly affects metamorphosis and also caused mortality, reported by Agüero et al. (2023).
In another study evaluating the effect of the insecticide lufenuron on the green stink bug Nezara viridula (Linnaeus 1758) (Hemiptera: Pentatomidae), Furiatti et al. (2009) found that this IGR at a concentration of 0.025% efficiently controlled nymphs, causing more than 80% mortality after 24 hours of contact with this product, result this similar to that found in our study. Study about ingestion of the product novalurom (2.1 mg/l) by Leptinotarsa decemlineata (Say, 1824) (Coleoptera: Chrysomelidae), Xu et al. (2017) observed deleterious effects of this insecticide on fourth-instar larvae, noting that the inhibition of chitin biosynthesis impaired the pupal stage and consequently the emergence of the adults, which is similar to the results obtained in this work, when fourth-instar nymphs of E. heros were exposed to this IGR.
According to Zantedeschi et al. (2017), the insecticide lufenuron at the recommended dose to control caterpillars (100 g a.i. ha -1 ) had a deleterious effect on the reproduction of E. heros, reducing the number of eggs produced per female, similar to what was observed this work for the same species in a greenhouse. Ávila & Nakano (1999) evaluated the effect of the IGR lufenuron on adult Diabrotica speciosa (Germar, 1824) (Coleoptera: Chrysomelidae) when a 0.033% solution of this product was sprayed on bean plants. Couples of D. speciosa fed bean leaves treated with this IGRs had minors fecundity and viability of the eggs produced than untreated couples (control). These authors also observed that the non-viable eggs showed embryonic development, but the larvae did not hatch, results that partially corroborate to those obtained with E. heros in this work.
The reproduction of Schistocerca gregaria (Foskal, 1775) (Orthoptera: Acrididae) was also affected after application of the insecticide lufenuron (50, 100, 200, 300, and 400 ppm) on fifth-instar nymphs, being observed spermatocyte disorganization as well as the loss of testicular tissue and the Golgi apparatus (Ghazawy, 2012). Another study carried out by Mansur et al. (2010) to verify the effects of different doses of lufenuron (7.5 and 15 μg) on female Rhodnius prolixus (Stal, 1859) (Hemiptera: Reduviidae), was observed a 30 to 50% reduction in the oviposition rate of this stink bug. The stink bug Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae), when exposed to lufenuron, had a reduction in the viability of eggs, but the fecundity was not affected (Evangelista et al., 2002). The same was observed in the present study, when the stink bug E. heros was submitted to a low dose of lufenuron.
The use of insecticides can often cause stressful situations, not only because they are considered toxic in high doses, but in some situations in low doses they can have stimulating or beneficial effects (Guedes et al., 2016); (Cutler, 2013). In the case of males throughout the mating period, they use mechanisms such as: sperm mobility, sperm storage, stimulation of ovulation or oviposition and protection of the eggs, which make it possible to increase the transfer of genetic matter (Avila et al., 2011); (Ge et al., 2010). Males of E. heros, when exposed to jas.ccsenet.org Journal of Agricultural Science Vol. 15, No. 7; under doses, exhibit the behavior mentioned above, and may have the ability to alter or manipulate the secretions of their accessory glands to increase their performance (Yu et al., 2012;Haddi et al., 2016) and they are also encouraged to walk further and find a greater number of females for mating (Gatehouse, 1997). On the other hand, females of E. heros, when exposed to sublethal dosages of some insecticides, may have an increase in their reproductive capacity (Harano, 2015;Haddi et al., 2016), because when exposed to stress conditions, they can use this mechanism to increase the perpetuation of the species (Vahed, 2017). The same was observed in our work when the females were exposed to the lowest dose of the insecticide lufenuron, where their oviposition capacity was not altered when exposed to the product.
Studies performed in the laboratory by Tail et al. (2010) with the grasshopper S. gregaria, to verify the influence of the ingestion of diflubenzuron (30 mg/ml), the authors found reduced weight of the ovaries after females of this species ingested this IGRs. In a study with adult of Lygus lineolaris (Palisot Beauvois, 1818) (Heteroptera: Miridae), which ingested a diet incorporated with novaluron (600 ppm), Catchot et al. (2020) observed a negative effect on oviposition rate, ovarian maturation, and hatching of eggs laid by this species. These results corroborate those obtained in this work when adults of E. heros were exposed to soybean leaves treated with novaluron at the highest dose tested, for egg viability, although the target species and exposure mode were different.
Ferreira Aguero et al. (2014) when testing the juvenile physiological insecticide pyriproxyfen (100 g a.i. ha -1 ) and diflubenzuron (800 g a.i. ha -1 ) on N. viridula nymphs observed that pyriproxyfen reduced fertility and decreased the percentage of hatched nymphs, while diflubenzuron, affected the formation and development of the female and male reproductive system of this species. The same authors also found that the eggs presented low viability or empty embryos due to embryo malformation, similar to the results verified for E. heros in the present work.

Conclusion
In summary, the results obtained in our work showed that the IGRs tested on E. heros nymphs and adults caused deleterious effects in the nymphal ecdysis process, resulting in deformation and mortality of insects. In addition to affecting the reproductive process in the adult phase, producing nonviable eggs (without embryo formation) and reducing the quantity of eggs produced. Thus, we can infer that the insecticides lufenuron, novaluron, and teflubenzuron, although recommended to control caterpillars in soybean, may also be a complementary alternative for the management of E. heros in this crop.