Acta agriculturae Slovenica, 120/1, 1–13, Ljubljana 2024 doi:10.14720/aas.2024.120.1.13479 Original research article / izvirni znanstveni članek Investigation of diflovidazin and fenpropathrin on two-spotted spider mite, Tetranychus urticae Koch, 1836 (Acari: Tetranychidae): population and interaction study Mobina ABBASI 1 , Mohammad GHADAMYARI 1, 2 , Azadeh KARIMI-MALATI 1 , Elaheh Shafiei ALAVIJEH 1 Received April 27, 2023; accepted February 21, 2024. Delo je prispelo 27. aprila 2023, sprejeto 21. februarja 2024. 1 Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran 2 Corresponding author, e-mail: mghadamyari@gmail.com Investigation of diflovidazin and fenpropathrin on two-spot- ted spider mite, Tetranychus urticae Koch, 1836 (Acari: Tet- ranychidae): population and interaction study Abstract: Tetranychus urticae is one of the most impor- tant pests of agricultural crops around the world. This research investigated the lethal effects of diflovidazin and fenpropathrin on different life stages of two-spotted spider mites, the interac- tion of binary mixture of these two compounds, and sublethal effects of diflovidazin on the deutonymphs under laboratory conditions. The Potter spray tower was used for the bioassay of acaricides on different life stages of T. urticae. The results showed that diflovidazin was effective on different develop- mental stages excluding female’s adults. Also, fenpropathrin showed toxicity on all life stages except eggs. LC 50 value and combination index (CI) of their mixture against deutonymph were 4.85 mg l -1 and 0.5 mg 1 -1 , respectively, which revealed a synergistic effect on T. urticae. Sublethal effects of LC 30 concen- tration of diflovidazin were evaluated on life table parameters of T. urticae. The value of the intrinsic rate of increase (r), the finite rate of increase (λ), and the net reproductive rate (R 0 ) sig- nificantly decreased in treated mites in comparison to control. These results suggested that diflovidazin could have significant roles in the control of T. urticae due to negative effect on popu- lation parameters as well as synergistic effect of binary mixtures of this acaricide with fenpropathrin. Key words: two-spotted spider mite, life table, mixture of pesticides, interaction Preučevanje učinkov diflovidazina in fenpropatrina na na- vadno pršico (Tetranychus urticae Koch, 1836) (Acari: Te- tranychidae): populacijska in interaktivna raziskava Izvleček: Navadna pršica (Tetranychus urticae) je eden izmed najpomembnejših škodljivcev gojenih rastlin širom po svetu. V raziskavi so bili preučevani letalni učinki diflovidazina in fenpropatrina na različne razvojne stopnje navadne pršice, učinek mešanic teh dveh snovi in subletalni učinki diflovidazi- na na deutonimfe v laboratorijskih razmerah. Za preučevanje učinkov teh akaricidov na različne razvojne stopnje navadne pršice je bil uporabljen Potterjev pršilni stolp. Rezultati so po- kazali, da je bil diflovidazin učinkovit v vseh razvojnih stopnjah, z izjemo odraslih samic. Tudi fenpropatrin je bil strupen za vse razvojne stopnje pršice, z izjemo jajčec. LC 50 vrednost in kom- binacijski indeks (CI) mešanic akaricidov na deutonimfe sta bila 4,85 mg l -1 in 0,5 mg 1 -1 , kar kaže na njun sinergijski učinek pri zatiranju navadne pršice. Vrednosti maksimalne rasti popu- lacije (r), končne rasti populacije (λ) in neto reprodukcije (R 0 ) so se pri obravnavanih pršicah značilno zmanjšale v primerjavi s kontrolo. Rezultati nakazujejo, da bi diflovidazin lahko imel pomebno vlogo pri nadzoru navadne pršice zaradi njegovih ne- gativnih učinkov na populacijske parametre kot tudi zaradi si- nergističnih učinkov mešanic tega akaricida s fenpropatrinom. Ključne besede: navadna pršica, preživetvena sposob- nost, mešanica pesticidov, interakcija Acta agriculturae Slovenica, 120/1 – 2024 2 M. ABBASI et al. 1 INTRODUCTION The two-spotted spider mite, Tetranychus urticae Koch, 1836 (Acari: Tetranychidae) is one of the most destructive polyphagous mites in the greenhouses and fields and causes significant damage to the yield of many horticultural, agricultural, and ornamental crops (Jepp- son et al., 1975). Nearly 1,200 plant species have been reported as hosts of this mite, of which more than 150 species of these hosts such as cotton, corn, tomatoes, and ornamental trees have economic importance. This mite can produce more than 37 generations per year under fa- vorable conditions (Riahi et al., 2013). The management of T. urticae populations has relied on application of dif- ferent chemical acaricides (Cooper & Dobson, 2007) that have increased the public health concern (Owusu & Yeboah, 2007) and adverse effects on beneficial organ- isms (Bajc et al., 2017; Eziah et al., 2016). Due to high reproductive ability and short life cycle, two-spotted spider mite could develop resistance to many acaricides (Devine et al., 2001; Stumpf & Nauen, 2001). Despite the discovery and commercialization of acaricides belonging to different chemical groups, the number of acaricides on the market is much lower than insecticides that were estimated 7 % of the total insecticides market (Sparks & Nauen, 2015). Therefore, it is important to prevent the resistance of acari mites to acaricides and using a mixture of pesticides is one of the ways to prevent pest resistance to pesticides. One of the resistance management programs is to use a mixture of pesticides for more effective control (Ahn et al., 1993). A mixture of two or more pesticides may improve pest control, enhance their pesticidal prop- erties, decrease the costs and pesticide consumption and prevent pesticide resistance (Yuya et al., 2009). However, the influence of this way depends on the amount of appli- cation and the formulation of the pesticides. Widespread use of pesticide mixtures is usually common in green- houses and seedling production centers where a collec- tion of pests are present (Hosseininaveh & Ghadamyari 2013). In addition, multi-pesticide mixtures usually alter the uptake, transport, metabolism, and toxicity at the target site of pesticides, thus improving performance in many cases (Flint, 2002). On the other hand, sublethal insecticide exposure is expected to increase the competi- tiveness of resistant phenotypes (Wang et al., 2018), act- ing as a selection pressure for the evolution of insecticide resistance (Hamedi et al., 2010). The effects of sublethal concentrations of acaricides have been studied on spi- der mites (Landeros et al., 2002; Marčić, 2005, 2007). To better estimation of pesticides, the investigation on life table and population fitness could clarify the impacts of pesticide on pests and non-target species. Thus eco toxi- cological improvement could associate with altering the way of the evaluation of pesticide effects (Kammenga et al., 1996; Stark & Banks, 2003). Different groups of pesticides are used for control of the tetranychid family in Iran (Saeidi & Arbabi, 2007). Fenpropathrin (Danitol ® ) is a pyrethroid insecticide used to control this pest (IRAC group 3A, sodium channel modulators) which was documented for reducing the rate of reproduction by feeding prevention and disrup- tive vital functions of adult mites (Nourbakhsh, 2019). Recently, diflovidazin (Flumite ® ) is a compound in the tetrazine chemical (IRAC group 10A, mite growth in- hibitors affecting CHS1) (Merzendorfer, 2013) has been registered for management of T. urticae and Panonychus ulmi (Koch, 1836) (Acari: Tetranychidae) in Iran (Nour- bakhsh, 2019). Therefore, in this study, the lethal effects of fen- propathrin and diflovidazin were assayed on different stages of two-spotted spider mite. After determining the LC 50 , the mixture of these compounds was examined for detection of synergistic, antagonistic, and potentiation effects on deutonymphs. Finally, the influence of LC 30 concentration of diflovidazin was evaluated on life-table parameters as population growth parameters, female longevity, and life expectancy of T. uticae. 2 MATERIALS AND METHODS 2.1 MITE POPULATION GROWTH AND CHEMI- CAL COMPOUNDS The mite population was collected from Siahkal (Gilan province, Iran) considered as susceptible to acari- cides, in the summer of 2016 without a history of apply- ing synthetic pesticides. The population was established on black-eyed pea (Vigna unguiculata (L.) Walp) under the laboratory conditions (25 ± 2 °C, 60 ± 10% relative humidity (RH), and a 16:8 h (L:D) photoperiod). Commercial formulations of fenpropatrin 10 % EC (Danitol ® ) and difluvidazin 20 % SC (Flumite ® ) were ob- tained from Kimia Gohar Khak and Agro-Chemie Ltd, respectively. 2.2 BIOASSAYS ON DIFFERENT DEVELOPMEN- TAL STAGES The toxicity status of pesticides was determined on each developmental stage of T. urticae (Vassiliou & Kitsis, 2013). Briefly, 10-15 same age larvae (0-24 hour old), deutonymph, and female adults were transferred Acta agriculturae Slovenica, 120/1 – 2024 3 Investigation of diflovidazin and fenpropathrin on two-spotted spider mite, ... on the upside of each leaf discs of black-eyed pea, Vigna unguiculata (L.) Walp. (4 cm 2 ). After mite’s settlement, different concentrations of fenpropathrin were sprayed on leaf discs containing deutonymph (4, 8, 16, 32 and 65 mg l -1 ), larvae (2, 4, 6, 10 and 16 mg l -1 ) and adult (10, 20, 40, 80 and 100 mg l -1 ). Also, different concentrations of difluvidazin were applied on egg (1, 2, 3, 5, 7 and 10 mg l -1 ), larvae (1, 2, 3, 5, 10 and 15 mg l -1 ) and deutonymph (1, 2.5, 5, 10 and 20 mg l -1 ) by a Potter spray tower (1 bar pressure, 1.5 ± 0.05 mg spray fluid deposit/cm 2 ). The dis- tilled water was used as control. Mortality was assessed 48 h after treatment. Mites that did not walk normally after touching with a camel’s hair brush were considered as dead. For the egg developmental stage, 24 hours before bioassay, 10 female adult mites were put on a not-treated leaf disc for oviposition. Then, 20 eggs were used for each replication. For each treatment, 5 replications were pre- pared. All sample for each bioassay were transferred to the controlled conditions in an incubator at 25 ± 2 °C, 60 ± 10 % RH, and a 16:8 h (L:D) photoperiod. 2.3 THE MIXTURE BINARY ASSAY OF FEN- PROPATHRIN AND DIFLOVIDAZIN Toxicity interaction studies were based on LC 50 val- ues for deutonymph stage of T. urticae. The mixture ratio was prepared based on LC 50 values of fenpropathrin and difluvidazine using the potter tower bioassay method as discussed above. So, the binary mixture effects of these two pesticides were evaluated in 1 (diflovidazin): 2 (fen- propathrin) ratios. As above, the bean leaf discs contain- ing deutonymph were sprayed with different doses (1, 2, 7, 14 and 20 mg l -1 ) of a mixture of two pesticides, and the value of LC 50 in the mixture was determined as described above. 2.4 DIFLUVIDAZIN SUBLETHAL EFFECT AND LIFE-TABLE ASSAY For evaluation of the sublethal effects of diflovi- dazin, the LC 30 concentration was investigated on deu- tonymph of two-spotted spider mite. In order for this assay, 100 deutonymphs were transferred on leaf discs and treated with LC 30 concentration. Distilled water was used as control. After being adults, 15 pairs of T. urticae were transferred on new leaf discs, separately, and their oviposition as well as mortality were documented until the death of all individuals. Moreover, same aged-eggs laid on leaf discs (within a 24-h for both treatments) were used for life history experiments. During this test, developmental times and mortality of different immature stages were checked daily until adult emergence. There- fore, the raw life history data of treated and untreated T. urticae were obtained. 2.5 STATISTICAL ANALYSIS LC 30 and LC 50 values, slopes, toxicity ratios between developmental stages in 95 % confidence intervals were determined by Probit analysis (POLO-PC, LeOra Soft- ware, Berkeley, USA). Furthermore, LC 50 of two pesti- cides and LC 50 of each pesticide on different life stages of two-spotted mite were compared by POLO-PC software and toxicity ratios were calculated to find the higher ef- fects (Robertson et al., 2017). Binary mixture of two acaricides were analyzed based on the method of Chou & Talalay, 1984. For this purpose, CompuSyn software was used which by cer- tain ratios of each compound, the combination index, CI was calculated and the type of interaction effect was detected as synergistic, additive, and antagonistic. The combination index (CI) has been used for the quantita- tive determination of synergism (CI < 1), antagonism (CI > 1), and additive effect (CI = 1), and CI is calculated by Compusyn software. The figures were designed by Microsoft Excel 16. The recorded raw data for life history and population growth parameters of T. urticae were analyzed based on the life table theory (Chi & Liu, 1985) and computer pro- gram TWOSEX-MS Chart (Chi, 2015). The age-specific survival rate (l x ), the age-stage-specific survival rate (s xj ), the age-specific fecundity (mx), the age-stage-specific reproductive values (v xj ), and population growth param- eters such as intrinsic rate of increase (r), finite rate of increase (λ), net reproductive rate (R 0 ) and mean genera- tion time (T) were calculated, accordingly. Moreover, the bootstrap method (100,000 replications) was used for the means and standard errors (Chi, 2015). The means were compared using the paired bootstrap test at 5 % signifi- cant level based on the confidence interval of difference (Efron and Tibshirani, 1993; Chi, 2015). 3 RESULTS AND DISCUSSION 3.1 EV ALUATION OF THE LETHAL EFFECT OF DIFLOVIDAZIN AND FENPROPATHRIN The LC 50 and LC 30 values of diflovidazin and fen- propathrin are presented in Table 1 and Table 2, respec- tively. Acta agriculturae Slovenica, 120/1 – 2024 4 M. ABBASI et al. The LC 50 values on the eggs, larvae, and deu- tonymphs indicated that the eggs had a higher suscepti- bility to diflovidazin. The greater slope of the logarithm- probit line in diflovidazin treatments was related to the egg stage that showed the lower increase in concentration caused more mortality in eggs in comparison to other de- velopmental stages (Figure 1). Diflovidazin did not have any toxicity on females up to 10000 ppm (Table 1). Be- sides, fenpropathrin had no lethal effect on eggs up to 10000 mg l -1 (Table 2). The larval stage has the highest sensitivity to fenpropathrin rather than deutonymph and female adult (Table 2 and Figure 2). According to results (Table 3), the larval stage has the same sensitivity to fen- propathrin and difluvidazine (Figure 3). However, larvae showed most sensitivity to these pesticides. The toxicity ratio comparison of LC 50 of three de- velopmental stages in diflovidazin treatment is shown in Table 3. Due to overlap of the diflovidazin LC 50 values of deutonymph and larval stage (1.16- fold), that involvement of number one between the high and low 95 % confidence limits. The toxicity ratio of fenpropathrin was found larva as the most sensitive stage. The various biological stages were as follows: larvae > deutonymph > female adults. The comparison of toxicity of diflovidazin and fenpropathrin on larval and deutonymph stages showed that there was not a significant difference in the larval stage (1- fold). However, the deutonymph stage had more sensitivity to diflovidazin with 2.11 folds more than fenpropathrin. Table 1: Diflovidazin bioassay on different developmental stages of Tetranychus urticae LC 50 (mg l -1 ) (95 % CI + ) LC 30 (mg l -1 ) (95 % CI + ) df Chi-square Slope ±SE N* Developmental stage 3.18 (2.38-3.83) 2.21 (1.42-2.83) 4 2.29 3.34 ± 0.57 280 Egg 5.65 (2.60-8.63) 3.29 (0.80-5.31) 4 4.50 2.23 ± 0.44 280 Larvae 6.59 (4.42-8.60) 3.93 (2.00-5.51) 3 1.01 2.34 ± 0.49 240 Deutonymph 10000 ** NC - - - 240 Adult *The number of mites were used in bioassays CI + : The upper and lower confidence interval at 95 % level NC: Not calculated ** : It was not possible to calculate the LC 50 value due to the phytotoxicity property Table 2: Fenpropathrin bioassay on different developmental stages of Tetranychus urticae LC 50 (mg l -1 ) (95 % CI + ) LC 30 (mg l -1 ) (95 % CI + ) df Chi-square Slope ± SE N* Developmental stage 5.65 (4.20-6.89) 3.95 (2.51-5.07) 3 1.11 3.38 ± 0.60 240 Larvae 13.92 (10.06-18.16) 7.94 (4.93-10.86) 3 1.02 2.15 ± 0.32 240 Deutonymph 35.51 (25.15-46.39) 20.04 (11.46-27.75) 3 1.78 2.11 ± 0.35 240 Female adults 10000 ** NC - - - - Egg *The number of mites were used in bioassays CI + : The upper and lower confidence interval at 95 % level NC: Not calculated ** : It was not possible to calculate the LC 50 value due to the phytotoxicity property Acta agriculturae Slovenica, 120/1 – 2024 5 Investigation of diflovidazin and fenpropathrin on two-spotted spider mite, ... Figure 1: Logarithm of concentration-probit relationship percentage of mortality of different developmental stages of Tetranychus urticae in response to diflovidazin Figure 2: Logarithm of concentration-probit relationship percentage of mortality of different developmental stages of Tetranychus urticae in response to fenpropathrin Figure 3: Logarithm of concentration-probit relationship percentage of mortality of different fenpropathrin, diflovidazin and their mixture on Tetranychus urticae Acta agriculturae Slovenica, 120/1 – 2024 6 M. ABBASI et al. In the study of clofentzine (IRAC group 10A), pro- pargite (IRAC group 12C), tetradifon (IRAC group 12D), etoxazole (IRAC group 10B), fenpyroximate (IRAC group 21A), amitraz (IRAC group 19), fenpropathrin, hexythiazox (IRAC group 10A), bromopropylate (un- known Mode of Action (MOA)), and fenazaquin (IRAC group 21A), the most effective compounds were reported hexythiazox and etoxazole, but fenpropathrin had the least effect on deutonymphs of Iranian population of T. urticae (Saeidi & Arbabi, 2007). The toxicity of difluvi- dazine on the larval stage of citrus red mite, Panonychus citri (McGregor, 1916) (Acari: Tetranychidae) (Gao et al., 2004) is 173.39- times higher than its adult stage which was consistent with the results obtained in this research. Since diflovidazin inhibits chitin synthesis (IRAC group 10A) less or no effective was observed on females. Based on the results of Marčić (2000), eggs had more sensitivity to diflovidazin and clofentzin acaricides in comparison to larva and deutonymph of T. urticae. The LC 50 value of diflovidazin on females of T. urticae was 2362.2 ppm (Havasi et al., 2018). Aveyard et al. (1986) reported the effectiveness of clofentzine acaricide on the egg, larval, and deutonymph of T. urticae without influence on adults. The egg stage had a lower LD 50 (0.16 ppm) than other stages which were consistent with the results of this study. Fenpropathrin had no effect on the two-spotted mite egg stage. Similar to the results of this research, propargite did not show any toxicity effect on the egg stage of this pest (Ashley et al., 2006). The acaricide activity of bifenazate and diazene was reported on all developmental stages of T. urticae and P. citri; while etoxazole and tebufenpyrad (IRAC group 21A) had no effect on adult comparison bifenaz- ate (Ochiai et al., 2007). In the survey of clofentezine, deltamethrin (IRAC group 3A), fenpyroximate, and hexythiazox, hexythiazox had the lower LC 50 on larvae of three laboratory strains of T. urticae (Nauen et al., 2001). 3.2 INTERACTION OF DIFLOVIDAZIN AND FEN- PROPATHRIN The mortality of adult female T. urticae in response to different concentrations of a combination of diflovi- dazin and fenpropathrin (in a ratio of 1: 2 of LC 50 values) were reported in Table 4. The CI of the binary mixture of diflovidazin and fenpropathrin was determined 0.5 which is in the range of 0.3 < CI < 0.7, according to Table 4, the effect of the mixture was synergistic. The combination toxicity of diflovidazin LC 50 with fenpropathrin LC 50 in a ratio of 1: 2 was tested on T. ur- ticae deutonymph. The LC 50 values obtained from the ef- Table 3: The comparison of diflovidazin and fenpropathrin toxicity on different developmental stages of Tetranychus urticae Treatment Developmental stage Toxicity ratio (95 % Confidence Interval) * Diflovidazin LC 50 of deutonymph/ LC 50 of larvae 1.16 0.75-1.80 LC 50 of deutonymph/LC 50 of egg 2.07* 1.42-3.01 LC 50 of larvae/LC 50 of egg 1.77* 1.21-2.61 Fenpropathrin LC 50 of deutonymph/ LC 50 of larvae 2.46* 1.70-3.55 LC 50 of adult/LC 50 of deutonymph 2.55* 1.69-3.84 LC 50 of adult/LC 50 of larvae 6.38* 4.32-9.11 Larvae 1.00 0.67-1.47 Deutonymph 2.11* 1.39-3.20 *The significant difference in Toxicity ratio: the upper and lower confidence at 95 % level Table 4: The toxicity index values of diflovidazin and fen- propathrin mixture on Tetranychus urticae Mortality percentage Combination Ratio (2:1) diflovidazin + fenpropathrin Combination Index (CI) 10 0.91 0.514 20 1.62 0.516 30 2.38 0.517 40 3.27 0.518 50 4.37 0.518 60 5.83 0.519 70 8.00 0.520 80 11.75 0.521 90 20.97 0.523 95 35.74 0.525 Acta agriculturae Slovenica, 120/1 – 2024 7 Investigation of diflovidazin and fenpropathrin on two-spotted spider mite, ... fect of the mixture of them had been reported in Table 5. Based on the data in these table, the combined mixture of these two acaricides on the deutonymph stage is more toxic than either acaricide alone. A comparison of the toxicity ratios of diflovidazin, fenpropathrin, and binary mixture of these compounds on deutonymph with 95 % confidence has been given in Table 6. If the confidence interval of LC 50 ratios in- cludes one, it indicates no significant difference between two treatments (Wheeler et al., 2006). Therefore, in this study, the LC 50 of diflovidazin did not show a significant difference with LC 50 of the pesticide mixture which was related to the overlap of their LC 50 confidence limits and involvement of number one between the 95 % confidence limits of LC50 ratios (Table 6). In the mixture of fenvalerate (IRAC group 3A) and some organophosphate pesticide, the most effective com- bination was fenvalerate and azinphos-methyl (IRAC group 1B) in a 1:1 ratio on T. urticae (Bruce Chapman & Penman, 1980). The result of this study showed the combination of diflovidazin and fenpropathrin, with dif- ferent mode of action as sodium channel modulator and growth inhibitor, respectively, had a synergistic effect on T. urticae control. Similar to the results obtained for the mixture of difluvidazine with fenpropatrin (pyrethroid) in this research, a synergistic effect was observed in a mixture of chlordimeform with some pyrethroid com- pounds (El-Sayed & Knowles, 1984). 3.3 THE EFFECT OF LC 30 CONCENTRATION OF DIFLOVIDAZIN ON T. urticae Developmental times (day) and longevity of the T. urticae are presented in Table 7. As shown, the length of the egg incubation period, as well as the protonymph du- ration, were significantly different in diflovidazin treat- ment and control (Table 7). Analysis of variance showed that the adult preovi- positional period (APOP) and total preovipositional pe- riod (TPOP) were not different in treatment and control (T able 8). The sub-lethal dose of diflovidazin significantly reduced the oviposition period of females, which was due to the reduction of female longevity in diflovidazin treat- ment (3.47 ± 0.25) in the comparison with the control (10.14 ± 3.5). Also, the number of eggs was significantly reduced by the treated females compared to the control. In the present study, the effect of LC 30 concentration of diflovidazin on fecundity, adult preoviposition period, total preoviposition period was calculated (Table 8). The intrinsic rate of population increase (r), the finite rate Table 5: Diflovidazin + fenpropathrin bioassay on deutonymph of Tetranychus urticae Developmental stage N* Slope ± SE Chi-square df LC 50 (mg l -1 ) Deutonymph 240 1.96 ± 0.26 0.89 3 4.85 (3.71-6.10) *The number of mites were used in bioassays Table 6: Comparison of toxicity of fenpropathrin, diflovidazin and their mixture on deutonymph of Tetranychus urticae Treatment Toxicity ratio Up - Down * Fenpropathrin / Diflovidazin 2.11* 1.39 - 3.20 Fenpropathrin/ Mixture 2.86* 1.90 - 4.32 Diflovidazin/ Mixture 1.35 0.89 - 2.07 *The significant difference in confidence at 95 % level Table 7: The effect of diflovidazin on different stage duration of Tetranychus urticae (mean ± SE) Treatment Egg Larvae Protonymph Deutonymph Pre-adult period Female longevity Life span Control 3.75± 0.44 1.76 ± 0.43 2.11 ± 0.39 2.21 ± 0.52 9.83 ± 0.91 10.14 ± 3.5 19.97 ± 6.01 Diflovidazin 4.02 ± 0.02 * 1.62 ± 0..05 1.83 ± 0.06 * 2.29 ± 0.11 9.76 ± 0.12 3.47 ± 0.25 * 13.22 ± 0.4 * *The significant difference in confidence at 95 % level Table 8 . The sub-lethal concentration effect of diflovidazin on the reproduction capacity of Tetranychus urticae (mean ± SE) Fecundity (egg/female) Oviposition period (day) TPOP*** (day) APOP** (day) Treatment 76.19 ± 5.64 10.97 ± 0.65 9.47± 0.16 0.0 ± 0.00 Control 16.39 ± 1.66* 3.64 ± 0.31* 9.71 ± 0.14 0.0 ± 0.00 Diflovidazin *The significant difference at 95 % level **APOP: Adult pre-oviposition period ***TPOP: Total pre-oviposition period Acta agriculturae Slovenica, 120/1 – 2024 8 M. ABBASI et al. of increase (λ), and the mean generation time (T) were significantly lower in diflovidazin. The net reproduction rate (R 0 ) was 7- folds lower than the control. Decrease in T value in diflovidazin treatment is probably due to shorter female longevity compared to the control treat- ment (Table 9). The age-stage specific survival rate (S xj ) indicates the probability that a newborn will reach any age and stage of life. The egg incubation period, larval duration, and the preoviposition period in LC 30 treatment of diflovidazin treatment were shorter than control (Figure 4). The age-specific fecundity of female adults (m x ), the age-specific maternity (l x m x ), and the age-specific surviv- al rate (l x ) in different diflovidazin treatments and control are shown in Figure 5. In these curves, the age-specific fecundity indicates the rate of reproduction of females at different ages, as the onset time and reproduction ter- mination in treatments. The age-specific survival rate (l x ) indicates a newborn egg will survive to x age. Accord- ing to the results, m x and l x in diflovidazin treatment have a sharp decline and the fertility and survival of female adults in this treatment decreased rapidly (Figure 5). Table 9: The sub-lethal concentration effect of diflovidazin on the life table parameters of Tetranychus urticae (mean ± SE) T (day) GRR R 0 λ (day -1 ) r (day -1 ) Treatment 14.455 ± 0.214 93.87 ± 5.764 47.24 ± 6.247 1.30 ± 0.012 0.266 ± 0.009 Control 12.177 ± 0.158* 3.035 ± 3.72* 7.21 ± 1.090* 1.176 ± 0.014* 0.162 ± 0.012* Diflovidazin *The significant difference at 95 % level r: Intrinsic Rate of Increase, λ: Finite Rate of Increase, R 0 : Net Reproduction Rate, GRR: Gross Reproduction Rate, T: Mean Generation Time Figure 4: Age-stage survival rate (s xj ) of Tetranychus urticae at different stages in control and diflovidazin treatments Acta agriculturae Slovenica, 120/1 – 2024 9 Investigation of diflovidazin and fenpropathrin on two-spotted spider mite, ... Age-stage-specific reproductive value (v xj ) repre- sents the number of offspring expected to be generated in the future by any person of x age and j growth stage (Carey, 1993; Fisher, 1958). There was a significant dif- ference in comparison between the control reproductive value curves and the adult stage treatment (Figure 6) and the highest amount of reproductive value occurred in the control treatment on days 9-10. The age-specific life expectancy (e xj ) was summarized in Figure 7. Life expec- tancy changes had an inverse relation to mortality rate (q x ) and it was the lowest in diflovidazin treatment. The acaricide sublethal effect measurements could clarify the different aspects of acaricides on mites. The sublethal effects of bifenazate (IRAC group 20D) affected the parental generation of T. urticae as survival rate, ovi- position period, fecundity, and longevity (Li et al., 2017). The study of diflovidazin sublethal effects on T. urticae was associated with a significant reduction in biological parameters as female maturation duration, the oviposi- tion period, the net reproductive rate (R 0 ), intrinsic rate of increase (r), and total fecundity (Havasi et al., 2018). Sublethal concentrations of tebufenpyrad significantly affected the offspring, longevity, fertility, and the intrinsic rate of increase (r) of T. urticae (Marčić, 2005). Figure 5: Age-specific survival rate (l x ), age-specific fecundity of the female (m x ) and age-specific maternity (l x m x ) of Tetranychus urticae at different stages in control and diflovidazin treatments Acta agriculturae Slovenica, 120/1 – 2024 10 M. ABBASI et al. Figure 6: Age-stage reproductive value (vxj) of Tetranychus urticae at different stages in control and diflovidazin treatments 4 CONCLUSION Although control of T. urticae has proven successful in many protected crops by some beneficial organisms, acaricides play a major role in control of this pest in field and greenhouse’s crops (Van Leeuwen et al., 2010). Con- sidering that the cost of chemical control is lower com- pared to biological control by releasing predatory mites in the field (Vidrih et al., 2020). This justifies the inten- sive use of acaricides in some areas. In addition to using biological control agents to control this pest, the mixture of pesticides should also be considered as a strategy to delay resistance. The results of diflovidazin studies on T. urticae showed that diflovidazin is an effective acaricide on immature stages especially eggs. However, it had no effect on the adult stage. Fenpropathrin had the most influence on the larval stage, without effect on the egg stage. The results of LC 30 of diflovidazin acaricide were associated with the significant reduction of net reproduc- tion rate (R 0 ), intrinsic population growth rate (r), and the finite increase in population (λ). Thus, the combina- tion of these pesticides with complication effects on to- gether and a synergistic effect on deutonymph, can be ef- fective recommendation in T. urticae control and suitable for delay to acaricide resistance. Acta agriculturae Slovenica, 120/1 – 2024 11 Investigation of diflovidazin and fenpropathrin on two-spotted spider mite, ... Figure 7: Age-stage life expectancy (e xj ) of Tetranychus urticae at different stages in control and diflovidazin treatments 5 ACKNOWLEDGMENTS The authors express their gratitude to the Research Council of the University of Guilan for financial support during the course. 6 REFERENCES Ahn, Y .-J., Kwon, M., Y oo, J.-K., & Byun, S.-J. (1993). 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