Acta agriculturae Slovenica, 118/4, 1–8, Ljubljana 2022 doi:10.14720/aas.2022.118.4.2519 Original research article / izvirni znanstveni članek Biological and biochemical effects of lufenuron on Xanthogaleruca luteo- la (Muller, 1766 ) (Coleoptera: Chrysomelidae) Bahareh MOHAMMADZADEH TAMAM 1, Mohammad GHADAMYARI 1, 2, Elaheh SHAFIEI ALAVI- JEH 1 Received January 20, 2022; accepted October 03, 2022. Delo je prispelo 20. januarja 2022, sprejeto 3. oktobra 2022 1 Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran 2 Corresponding author, e-mail: ghadamyari@guilan.ac.ir Biological and biochemical effects of lufenuron on Xantho- galeruca luteola (Muller, 1766 ) (Coleoptera: Chrysomelidae) Abstract: Xanthogaleruca luteola (Mull., 1766) is the ma- jor defoliator pest of elm trees in urban area. In this study the effect of lufenuron on some biochemical and biological char- acteristics was investigated on X. luteola. The LC30 and LC50 of lufenuron were determined on the second instar larvae as 20.22 and 36.65 mg l-1, respectively. Effects of LC30 and LC50 concen- trations of lufenuron on some biological parameters showed that lufenuron caused an increase in larval, pre-pupal and pu- pal developmental periods. Also, none of the female insects that emerged from the treated larvae did not spawn during their life. The LC50 concentration of lufenuron decreased carbohydrate, lipid and protein content and increased glycogen content. But there was not a significant difference in glycogen, and protein contents following the exposure to LC30 concentration. How- ever, glutathione-s-transferase (GST) and esterase activities were significantly increased at LC50. In conclusion, due to lethal and sublethal effect of lufenuron on biochemical and biological traits of X. luteola, it can be recommended for control this pest in IPM program. Key words: Xanthogaleruca luteola; lufenuron; develop- mental periods; sublethal effects; biochemical parameters Biološki in biokemični učinki lufenurona na hrošča Xantho- galeruca luteola (Muller, 1766) (Coleoptera: Chrysomelidae) Izvleček: Hrošč Xanthogaleruca luteola Mull je najpo- membnejši defoliator brestov v urbanem okolju. V raziskavi so bili preučevani učinki lufenurona na nekatere biokemične in biološke lastnosti tega hrošča. LC30 in LC50 lufenurona sta bili določeni na drugem razvojnem štadiju ličink in sicer 20,22 in 36,65 mg l-1. Učinki LC30 in LC50 koncentracij lufenurona na nekatere biološke parametre so pokazali, da je lufenuron pov- zročil povečanje razvojnih obdobij ličinke, obdobja pred zabu- bljenjem in obdobja bube. Nobena od samic, ki so se izlegle iz obravnavanih ličink v celotnem življenskem obdobju ni odlegla jajčec. Koncentracija LC50 je zmanjšala vsebnost ogljikovih hi- dratov, maščob in beljakovin ter povečala vsebnost glikogena, ni pa bilo značilnih razlik v vsebnosti glikogena in beljakovin pri izpostavitvi. LC30 koncentraciji. Aktivnosti glutation-s- -transferaze (GST) in esterase sta se pri izpostavitvi LC50 značil- no povečali. Zaključujemo, da bi zaradi letalnih in subletalnih učinkov lufenurona na biokemične in biološke lastnosti tega hrošča to sredstvo lahko priporočili za uravnavanje škodljivcev in v programih integriranega uravnavanja škodljivcev. Ključne besede: Xanthogaleruca luteola; lufenuron; ra- zvojna obdobja; subletalni učinki; biokemični parametri Acta agriculturae Slovenica, 118/4 – 20222 B. MOHAMMADZADEH TAMAM et al. 1 INTRODUCTION The elm leaf beetle, Xanthogaleruca luteola (Muller, 1766) (Coleoptera: Chrysomelidae), is one the most de- structive pests of elm trees in Iran. This beetle in both larval and adult stages by feeding on the elm leaves (Ul- mus spp.) causes severe injurious to trees. In addition to defoliation and morphological changes, this pest causes physiological stress which increases the elm suscepti- bility to secondary pests and diseases. Additionally, X. luteola transmits the fungi spores of Dutch elm disease, Ophiostoma (Ceratocystis) novo-ulmi Brasier, that as- sumption the serious menace to these trees (Huerta et al., 2010). Due to the widespread plantation of elm trees in urban areas, the application of pesticides against X. lu- teola poses some problematic side-effects on human so- cieties. Therefore, the application of pesticides with high selectivity to this pest and low toxicity to humans and environment is highly appreciated (Defagó et al., 2006). The estimation of insecticide effects are accessed by lethal and sublethal studies through mortality assays and observation of biology, physiology, behavior and demo- graphic aspects of insect pests and natural enemies(De França et al., 2017). Among the insecticides, insect growth regulators (IGRs) seem to have most adverse ef- fects on insect pests. IGRs may affect the development of insect pests by the interruption of the molting pro- cess and cuticle formation, as well as disruption in the endocrine system of insects (Desneux et al., 2007). IGR compounds play a crucial role in control of insect pests, especially pests associated in urban area. Because of spec- ificity in their mode of action and safety to humans, wild life and the environment, these compounds are suitable for pest control than other synthetic insecticides (Tunaz & Uygun, 2004). Chitin synthesis inhibitors is categorized as IGR which have been detected for controlling of wide vari- ety of immature insect pests (Tunaz and Uygun, 2004). Lufenuron (IRAC group 15) is a benzoylureas that classi- fied as an inhibitors of chitin biosynthesis affecting chitin synthase 1 on insects (Dhadialla et al., 2009; IRAC, 2020) which has been successfully effective against pest species from Lepidoptera, Coleoptera, Hemiptera, Diptera and Thysanoptera (FAO, 2008) due to larvicidal effect along with transovarial–ovicidal and ovicidal effects (Yasir et al., 2019; Abdel Rahman, 2017). Based on results of Ar- ruda et al. (2020), resistance inheritance to lufenuron was incompletely recessive, autosomal, and monogenic in Plutella xylostella (Linnaeus, 1758) (Lepidoptera: Plu- tellidae). Due to autosomal and recessive nature, resist- ance to this compound spreads at low rate lufenuron was registered against lepidopteran and psylla in Iran (Nour- bakhsh, 2019). The increasing in larval, pre-pupal and pupal devel- opmental periods were reported in lufenuron affection in sublethal treatment of Glyphodes pyloalis Walker, 1859; which was associated reduction in fecundity and fertility of female adults (Piri Aliabadi et al., 2016). The reduction of glucose, protein and carbohydrate contents has been reported in IGRS treatments of Pectinophora gossypiella (Saunders, 1844) (Lepidoptera: Gelechiidae) (Kandi et al., 2012). Toxicity and sublethal effects of lufenuron on elm leaf beetle has not been studied; besides, based on the mode of action of this pesticide, it seems that this com- pound is appropriate for control of this pest. The ob- jective of this research was evaluation of the toxicity of lufenuron on X. luteola. Subsequently, some biological and biochemical parameters on 2th instar larvae were di- rected at LC30 and LC50 levels. 2 MATERIAL AND METHODS 2.1 CHEMICALS Lufenuron (Match®, EC 50) was prepared from Syngenta Crop Protection (Iran, https://www.syngenta. ir/product/crop-protection/insecticide/match). Other chemical materials were purchased from Merck (Darm- stadt, Germany), Wako (Tokyo, Japan) and Fluka (Buchs, Switzerland). 2.2 LABORATORY MASS CULTURE OF X. LU- TEOLA The elm leaf beetle adults were collected from Uni- versity of Guilan campus (Rasht, Guilan province, Iran) without history of pesticide application. Insects were reared under laboratory conditions on the elm leaves at 25 ± 2 °C, 16:8 photoperiod (L:D) and 75 % relative hu- midity (RH). Transparent plastic jars (10 cm × 7 cm × 5 cm) containing holes in the lids were used for the rearing of larvae and adults. In order to obtain larvae in the same age for bioassay tests, each pair of male and female adults was kept in similar plastic jars and the laid eggs were put in a new container (14 cm × 12 cm × 5 cm) in laboratory condition as mentioned above, daily. 2.3 BIOASSAY Bioassay tests were carried out on 2th instar larvae based on leaf-dip method (Memarizadeh et al., 2011). Five concentrations (10, 17.78, 31.62, 56.23 and 100 mg Acta agriculturae Slovenica, 118/4 – 2022 3 Biological and biochemical effects of lufenuron on Xanthogaleruca luteola (Muller, 1766 ) (Coleoptera: Chrysomelidae) l-1) of lufenuron were used for determination of LC30 and LC50 which were diluted in distillated water. Elm leaf discs (3 cm × 3 cm) were dipped in the desired concentrations for 30 seconds and dried at room temperature for 30 min before being offered to X. luteola larvae. The distillated water was used as control. Ten 2th instars were transferred to each plastic container containing treated leaf. Five replications were used for each treatment. Mortality was recorded 72 h after treatment. The LC30 and LC50 values were calculated using the Polo-PC software (Software, 1987). 2.3.1 Sublethal and lethal assays The evolution of sublethal and lethal effects of lufenuron in LC30 (20.22 mg l -1) and LC50 concentrations (36.65 mg l-1) were studied on 2th instar larvae with leaf- dip method. Larvae were fed on treated leaves for 48 h. Then, alive larvae were transferred to plastic jars which were nourished with fresh leaves up to adult emergence. During this test, mortality, larvae duration, pre-pupal duration, pupal duration, and fecundity of female adults were recorded. 2.3.2 Biochemical assay 2.3.2.1 Amounts of carbohydrate, lipid, and glycogen Biochemical assays were carried out on treated 2th instars with LC30 or LC50 doses of lufenuron. After 48 h, the whole body of surviving larvae were homogenized in sodium sulphate buffer solution (Na2SO4 2 %) for deter- mination of carbohydrate (Singh & Sinha, 1977), lipid and glycogen contents (Yuval et al., 1998). 2.3.2.2 Esterase activity measurement Esterase activity (Van Asperen, 1962) and glu- tathione-s-transferase (GST) activity (Habig et al., 1974) were measured based on using α- and β-naphthyl acetate (NA) and 1-choloro-2,4-dinitrobenzene (CDNB) as sub- strates, respectively. Three to four replicates were con- ducted for all previously mentioned enzyme assays. Protein concentration was measured according to Bradford (1976) method with bovine serum albumin as standard. 2.4 DATA ANALYSIS All statistical analyses were performed using SAS software (p ≤ 0.05). Tukey’s test statistic was used as com- parison means (Rodenhouse et al., 2004). 3 RESULTS The LC30 and LC50 values were determined as 20.22 and 36.65 mg l-1, 72 h after treatment, respectively which presented in Table 1. These concentrations were used as lethal and sublethal concentrations for the following ex- periments. 3.1 SUBLETHAL EFFECTS OF LUFENURON ON BIOLOGICAL PARAMETERS 3.1.1 Developmental periods The larval developmental duration was increased by 13.38 % and 27.06 %, when the larvae treated with LC30 and LC50 concentrations, respectively. Also, LC30 and LC50 concentrations were increased the pre-pupal by 13.2  % and 17.5 % and pupal by 16.65 % and 26.74 %, respec- tively. Totally developmental periods were significantly increased at LC30 and LC50 concentrations in comparison to the control which have been reported in Table 2. The investigation on female fecundity showed that emerged females from the treated 2th instar larvae did not oviposit any eggs during their lifetime have been lasted 10 days. 3.2 SUBLETHAL EFFECTS OF LUFENURON ON ENERGY RESERVES Significant differences were observed in carbohy- drate and lipid contents of the larvae in LC30 concentra- tion of lufenuron in comparison to the control which was significantly decreased 29.1 % and 45.44 %, respectively. However, protein and glycogen contents in this suble- thal concentration showed non-significant differences in comparison controls. The protein content was decreased by 12.38 % and glycogen content was increased signifi- cantly by 17.79 % (Table 3). Concentration (mg l-1) CL* LC30 20.22 14,71-25,93 LC50 36.65 29,45-46,38 Table 1: Determination of sublethal and lethal concentrations of lufenuron on 2th instar larvae of Xanthogaleruca luteola *CL (confidence limits) which has been calculated with 95 % confidence Acta agriculturae Slovenica, 118/4 – 20224 B. MOHAMMADZADEH TAMAM et al. The LC50 treatment was associated with significantly decreasing carbohydrate, lipid, and protein contents in comparison with untreated larvae, 27.8 %, 60.37 %, and 24.9 %, respectively, while glycogen content was signifi- cantly increased by 45.56 % (Table 3). 3.3 SUBLETHAL EFFECTS ON DETOXIFICATION ENZYME 3.3.1 Total esterase activity The esterase activity was increased by LC50 concen- tration 52.16  % and 62.75  %, respectively; when α-NA and β-NA used as substrates. Whereas, there were no significant differences between LC30 concentration and control (Table 4) 3.3.2 GST activity The GST activity were increased by 14.64  % and 69.71 %, when treated by LC30 and LC50 concentrations, respectively, which was significant at LC50 (Table 4). 4 DISCUSSION Investigation on sublethal effects of insecticides might be associated with variations in life history char- acteristics as growth developmental stages, fecundity, fertility (Stark & Banks, 2003; Saber et al., 2013; Rehan & Freed, 2015; Su et al., 2022), in addition to behavioral and physiological disturbances (Desneux et al., 2007). In this study, bioassay results showed that lufenuron is effective against X. luteola and LC50 was determined as 36.6 mg l -1. The results of present study showed that LC50 and LC30 concentrations had the considerable effects on the devel- opmental stages and fecundity of emerged female adults of X. luteola. Sublethal concentrations of lufenuron in- creased developmental stages in larvae, pre-pupal and pupal after the 2th instar larvae treated with LC30 and LC50 concentrations which were longer in LC50 concentration. This result is in consistent with the results of Kandi et al. (2012) which showed that lufenuron in LC50 con- centration increased the larval and pupal durations in Pectinophora gossypiella. Besides, the reducing in the adult longevity, fertility and pupal weight of Anticar- sia gemmatalis Hübner, 1818 (Lepidoptera: Noctuidae) was reported in the sublethal treatment of lufenuron, methoxyfenozide, spinosad, endosulfan, novaluron and Treatment Carbohydrate (mg/Larvae) ± SE* Protein (mg/Larvae) ± SE** Glycogen (mg/Larvae) ± SE Lipid (mg/Larvae) ± SE Control 77.7 ±2.2a 71.9 ± 2.1a 66.1 ± 6.8b 603 ± 2.8a LC30 55.1 ± 1.6 b 63 ± 0.8ab 80.4 ± 1.6b 329 ± 14.2b LC50 56.1 ±1.6 b 54 ± 2.4b 121.4 ± 3.6a 239 ± 12.8c Table 3: Effects of lufenuron on energy reserve of 2th instar larvae resulting from treated second instar larvae of Xanthogaleruca luteola *Means followed by the same letter do not differ significantly (p ≤0.05). **SE: Standard Error The carbohydrate contents (F = 19.61, df = 2,6, p value = 0.0023) The lipid contents (F = 286.11, df = 2, p value = 0.0004) The protein content (F = 6.93, df = 2,6, 11, p value = 0.028) The glycogen content (F = 1.24, df = 2,6, p value = 0.354) Treatment larval developmental duration (3th instar larvae) (day) ± SE* Pre-pupal duration (day) ± SE** Pupal duration (day) ± SE Fecundity (%) Control 6.93 ± 0.06c 2.17 ± 0.06c 6.96 ± 0.08c 41.01 LC30 8± 0,08 b 2.5 ± 0.12a 8.35 ± 0.19b 0 LC50 9.5 ± 0.12 a 2.63 ± 0.15a 9.5 ± 0.18a 0 Table 2: Life stages duration of Xanthogaleruca luteola after treatment with lufenuron *Means followed by the same letter do not differ significantly (p ≤ 0.05). **SE: Standard Error Larval developmental duration (F = 421.86, df = 2, 66, p value < 0.0001) Pre-pupal duration (F = 9.88, df = 2,53, p value = 0.0002) Pupal duration (F = 62.54, df = 2,45, p value < 0.0001) Acta agriculturae Slovenica, 118/4 – 2022 5 Biological and biochemical effects of lufenuron on Xanthogaleruca luteola (Muller, 1766 ) (Coleoptera: Chrysomelidae) tebufenozide (Storch et al., 2007). Also, the delay in the developmental duration of Cotesia flavipes (Cameron, 1891) (Hymenoptera: Braconidae) in parasitizing of Dia- traea flavipennella (Box, 1931) (Lepidoptera: Crambidae) was observed in sublethal affection of lufenuron (Fonse- ca et al., 2015). Evaluation of flufenoxuron on biological parameters was associated with increasing the larval and pupal periods and morphogenic abnormalities in devel- opmental stages of Spodoptera littoralis (Boisduval, 1833) (Reda et al., 2010). In this study, the fecundity and reproduction of X. luteola was influenced by lufenuron. Females that emerged from the treated larvae with LC30 and LC50 doses of lufenuron did not lay any eggs during their lifetime. The effect of lufenuron on fertility has been attributed to morphological changes in the ovipositor, interference with vitellogenesis, testicular size reduction, and sperm transport incapacity (Sáenz-de-Cabezón et al., 2006). Decreased fertility in IGR-treated insects may be associ- ated with IGR intervention in egg protein accumulation, vitellogenesis synthesis, uptake, and ovariole growth (Pineda et al., 2007). The results are accordance with the reduction of oviposition period in treatment with lufenu- ron (Josan & Singh, 2000), and cantharidin, selective in- hibitor of protein phosphatase 2A (Zhang et al., 2003), has been reported on Plutella xylostella (L., 1758) (Huang et al., 2015). Hexaflumuron decreased the oviposition pe- riod, egg numbers, and adult emergence of P. xylostella (Mahmoodvand et al., 2012). Embryonic development changes have been reported in azadirachtin, lufenuron and deltamethrin sublethal treatments on Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae) (Correia et al., 2013). In addition, fecundity declined in LC50 value of lufenuron on Heticoverpa armigera (Hub- ner, (1808)) (Butter et al., 2003). The percentage of egg hatching was reduced as a dose-dependent manner when S. littoralis treated with flufenoxuron (Reda et al., 2010). On the other hand, the exposure to insecticide sublethal concentrations could influence the biologi- cal, physiological and biochemical parameters such as carbohydrates, lipids, and proteins content (Klowden, 2013). When an insect is treated with insecticide, high level energy consumption occurs during detoxification of insecticides. This phenomenon may be leads to lower or higher larval duration or a reduction in reproductive performance (Boivin et al., 2001) which is evidence also in present results. Carbohydrates are assumed as the basic source of energy and a starting material in chitin synthetase (Genc et al., 2002; Nation, 2008). Further- more, lipids have major roles in preparation of energy, metamorphosis, exoskeleton substrates and biosynthesis of pheromones (Nation, 2008). Proteins are involved in structural and enzymatic functions as hormones and en- zymes biosynthesis which are able to convert as an en- ergy source (Klowden, 2013; Wigglesworth, 2012). Nu- tritional deficiencies along with the increase in metabolic activities for detoxification process during the exposure to pesticides are among the main reasons for the reduced energy level (De Coen & Janssen, 1997; Verslycke et al., 2003). Our results showed that a decrease in the level of energy sources, carbohydrate, lipid and protein, of the larvae following their treatment with LC30 and LC50 val- ues of lufenuron. According to these results, the protein contents of X. luteola larvae decreased at both LC30 and LC50 treatments, however there was only significant dif- ference in LC50 concentration compared to the control. This reduction could be due to the breaking the proteins into amino acids and their entry into the tricarboxylic acid (TCA) cycle as keto acid (Schoonhoven, 1982) to compensate for lower energy caused by lufenuron stress. The present results are in agreement with those of Kan- di et al. (2012) who reported LC50 of lufenuron caused reduction in the total soluble protein. Piri Aliabadi et al. (2016) showed a reduction in the protein content of Glyphodes pyloalis Walker, 1859 larvae when treated with LC30 and LC50 of lufenuron. The exposure to pesticides can affect carbohydrate metabolism in different species of insects by either decreasing or increasing its content Treatment α-Esterase (µmol.min-1.mg protein-1) ± SE* β-Esterase (µmol.min-1.mg protein-1 ) ± SE** GST (µmol.min-1.mg protein-1 ) ± SE Control 367.537 ± 14.496b 36.703 ± 0.903b 6.876 ± 1.593b LC30 442.370 ± 84.631 b 55.084 ± 3.915b 8.055 ± 1.004b LC50 768.221 ± 34.693 a 98.506 ± 10.387a 22.7 ± 2.9a Table 4: Effects of lufenuron on enzyme activities of 2th instar larvae resulting from treated second instar larvae of Xanthogaleruca luteola *Means followed by the same letter do not differ significantly (p ≤ 0.05). **SE: Standard Error The α-NA activity (F = 15.88, df = 2.6, p value = 0.004) The β-NA activity (F = 26.55, df = 2.6, p value = 0.0124) The GST activity (F = 19.41, df =2 , p value = 0.0192) Acta agriculturae Slovenica, 118/4 – 20226 B. MOHAMMADZADEH TAMAM et al. (Mansingh, 1972). In this study, carbohydrate content of the elm leaf beetle larvae dropped when they were treat- ed with lufenuron. Kandi et al. (2012) had observed the same results by using lufenuron against Pectinophora gos- sypiella (Saunders). Results of this study demonstrated a significant decrease in the lipid content of the larvae of the elm leaf beetle following their exposure to LC30 and LC50 concentrations of lufenuron. According to some studies, the exposure to pesticides affects synthesis and storage of lipids more than their breakdown (Ali et al., 2011; He et al., 2020). A similar result was reported for G. pyloalis larvae treated with lufenuron (Piri Aliabadi et al., 2016). Bashari et al. (2014) also showed a decrease in the lipid content of X. luteola larvae treated with hexaflumu- ron. Glycogen is one of the essential nutrient reserves in insect which can also be affected by pesticide treatment (Fahmy & Dahi, 2009). Our results revealed a significant increase in the glycogen level of the X. luteola when treat- ed with LC50 concentration of lufenuron. Changes in gly- cogen level may be due to disruption of the homeostasis mechanism in insects (Nath, 2002; Oguri & Steele, 2007). Lufenuron significantly reduced larval and pupal mass and extended duration of both developmental stages of Helicoverpa armigera (Hübner) (Lepidoptera: Noctui- dae) in LC90, LC50 and LC10 concentrations (Butter et al., 2003). The detoxification enzymes including monooxyge- nases, GST and hydrolases play the important roles in insecticide metabolisms (Yu, 2014). Additionally, im- provement of insecticide tolerance in short time and in- secticide resistance after a long period of time exposure may even incorporated by increasing in specific activi- ties and much expression of metabolic enzymes (Yin et al., 2008). Results of our study showed an increase in α- and β-esterase activities of the treated larvae which was significant at LC50 and not significant at LC30 compared to the control. In lufenuron treatment on H. armigera, the esterase activity was reduced significantly with cor- relation to dose- and time-dependent manner compared with control. It has been recommended the modification in esterase enzyme activities could have important role in fecundity and fertility reduction and inhibition of meta- morphosis (Reda et al., 2010). GSTs are another group of detoxifying enzymes which play an important role in the physiology of stress and intracellular transport and biosynthesis pathways of different cycles (Wilce & Parker, 1994). Results of this study showed that GST activity was increased after the treatment of the larvae with LC50. This result demon- strates that GST activity of the elm leaf beetle maybe are involved in the detoxification of lufenuron as detoxifica- tion enzyme for conjugating pesticides and their metabo- lite with glutathione. Bashari et al. (2014) also reported enhanced activities of GST in X. luteola after hexaflumu- ron treatment. 5 CONCLUSION Lufenuron in lethal and sublethal concentrations (LC50 and LC30) caused affections on the developmental, survival and fecundity of the second instar larvae of X. luteola that has been significant influences. In practi- cal approach, these impacts could modify the offspring numbers and maintain population under economic threshold level (ETL). 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