Sublethal Effects of Two Insecticides, Deltamethrin, Thiamethoxam and the Botanical Insecticide ( Foeniculum vulgare Mill.) on Callosobruchus maculates (Fabr.) (Coleoptera: Bruchidae)

The cowpea beetle Callosobruchus maculates (Fabr.) is a serious pest that infests pulse seeds in storage. The current study aimed to assess the sublethal effects of two insecticides deltamethrin, thiamethoxam and a botanical power of fennel ( Foeniculum vulgare Mill) on C. maculatus development and its essential enzymes. Thiamethoxam, deltamethrin insecticides and fennel plant powder activities against the cowpea beetle, C. maculatus, were assessed throughout direct contact application. Thiamethoxam showed the LC 50 value of 21.05 ppm followed by deltamethrin (LC 50 = 85.1 ppm) while, the least effect was recorded with fennel seeds powder with LC 50 value 36325.8 ppm. Concerning sublethal effects, sublethal concentrations of thiamethoxam (LC 10 , LC 25 , LC 50 and LC 75 ) significantly reduced egg-laying. While LC 75 of deltamethrin significantly decreased egg-laying related to control. In contrast, fennel seeds powder failed to turn down the number of eggs laid by C. maculatus females compared with the control. Complete protection to deltamethrin against C. maculatus adults was maintained after 24 h till 7 months and to thiamethoxam from 24 h till 4 months. Biochemical studies showed that the activity of acetylcholine esterase (AChE), α, ß esterase and glutathione -S-transferase (GST) showed a significant decrease in sublethal concentrations of thiamethoxam and deltamethrin related to the control. Regarding the effect on germination, no adverse effect was noticed due to the chemical and botanical seed protectants application.

The cowpea beetle Callosobruchus maculates (Fabr.) is a serious pest that infests pulse seeds in storage. The current study aimed to assess the sublethal effects of two insecticides deltamethrin, thiamethoxam and a botanical power of fennel (Foeniculum vulgare Mill) on C. maculatus development and its essential enzymes. Thiamethoxam, deltamethrin insecticides and fennel plant powder activities against the cowpea beetle, C. maculatus, were assessed throughout direct contact application. Thiamethoxam showed the LC50 value of 21.05 ppm followed by deltamethrin (LC50= 85.1 ppm) while, the least effect was recorded with fennel seeds powder with LC50 value 36325.8 ppm. Concerning sublethal effects, sublethal concentrations of thiamethoxam (LC10, LC25, LC50 and LC75) significantly reduced egglaying. While LC75 of deltamethrin significantly decreased egg-laying related to control. In contrast, fennel seeds powder failed to turn down the number of eggs laid by C. maculatus females compared with the control. Complete protection to deltamethrin against C. maculatus adults was maintained after 24 h till 7 months and to thiamethoxam from 24 h till 4 months. Biochemical studies showed that the activity of acetylcholine esterase (AChE), α, ß esterase and glutathione-Stransferase (GST) showed a significant decrease in sublethal concentrations of thiamethoxam and deltamethrin related to the control. Regarding the effect on germination, no adverse effect was noticed due to the chemical and botanical seed protectants application. So, the current work provides some information about the susceptibility of C. maculatus to deltamethrin, thiamethoxam insecticides and fennel (F. vulgare) as a botanical insecticide, assesses their sublethal effects on insect development, various reproductive activity parameters (fecundity and hatchability percentage) and critical enzyme activities such as AchE, CarE and GST. Furthermore, the present study was carried out to investigate the viability of faba bean (in terms of seed germination) post-treatment.

Chemicals and Insecticides Tested:
Two commercial formulations of thiamethoxam (Actara 25% WG, Syngenta) and deltamethrin (2.5% WP, Kafr El-Zayat Pesticides and Chemicals Company) and fennel seeds F. vulgare were purchased from an herb shop, Cairo, Egypt. Seeds were ground well in an electric mill, sieved using a 300-mesh sieve, and kept in tightly clean plastic jars and kept in the freezer.

Insect Cultures:
Cultures of the cowpea beetle, C. maculatus (F.) were reared on faba bean (variety Giza 2) at 27 °C and 65 ± 5% relative humidity (RH) in the Stored Grains pest Research Department, Plant Protection Research Institute, Agriculture Research Center. Before each trial, faba bean was fumigated with phostoxin tablets for at least three days to exclude infestation by insects.

Bioassays:
To estimate the insecticidal efficiency of thiamethoxam, deltamethrin and fennel seeds powders against C. maculatus adults. A sample of 10 g of fresh faba bean seeds was placed in a glass tube (1x7.5 cm) and exposed to five concentrations of the tested powders and then shake manually for one minute to achieve equal distribution of the powders and uniform coating of faba bean seeds with the tested material. Twenty-five C. maculatus adults (1 to 2 days old) were collected randomly from the stock colony and introduced into each prepared tube containing treated and untreated grains (control). Each experiment and the control were replicated 3 times. The tubes were covered with a secured plastic cover and maintained in the incubator. Mortality was assessed after 24 h post-exposure in the treated and untreated tubes and corrected according to Abbott formula (Abbott 1925) for natural mortality in controls.

Effect on Some Biological Aspects of C. maculatus:
To determine the number of eggs laid by mated female adults of C. maculates exposed to each of the tested materials, glass tubes measuring 1 × 7.5 cm contained 5 gm of cleaned faba bean seeds treated with LC10, LC25, LC50 and LC75 (separately) of each tested material (each treatment had three replicates), two pairs of newly emerged adults were placed in each tube and covered with plastic cover and kept in the incubator at 28 ±2 °C and 75±5% RH till death. The number of eggs laid was counted and the reduction in hatchability was determined.

Percentage of Adult Emergence:
The infested treated seeds with eggs were incubated and observed regularly until adult emergence. Emerging adults were counted daily for 10 days. The reduction percentage of emerging adults also was recorded.

Residue Determination:
Tubes measuring 1x7.5 cm each containing 10 gm of faba bean seeds treated with LC95 of each treatment and were divided into several groups. Each group consisted of three replicates. The tubes were kept under laboratory conditions just after mixing the faba bean seeds with tested powders. Treated seeds were stored for 14 months. Every month twentyfive adults of C. maculatus were placed on each of the three replicates, to show the residual effect of the treatments on the treated faba bean seeds. The tubes were covered with a plastic cover fixed with a rubber band, and the tubes were kept in laboratory conditions. Mortality counts were carried out after 24 h of introducing insects. Similar three replicates of untreated faba bean seeds were used as control. Activity of Enzymes: 1. Sample's Preparations: The untreated and treated adults (LC10, LC25, LC50 and LC75 values of actara, deltamethrin as ppm and fennel seeds) were transferred to a clean jar and kept at -40 °C, fifteen insects were weighed and homogenized in ice-cold of 0.1 M phosphate buffer with different pH values according to the enzyme in ratio 1:10 (W: V), five replicates were used to record the mean enzyme activities after 24 h from exposure.

Acetylcholine Esterase (AChE) Activity:
Acetylcholine esterase (AChE) activity was determined as indicated by Ellman et al. (1961) with modifications. Treated and untreated insects were homogenized in ice-cold 0.1 M phosphate buffer (pH 7.5) containing 0.1% (V/V) Triton X-100 then centrifuged at 13,000g at 4 ⁰C for 15 min. The supernatants were transferred to new tubes. 800 μl potassium phosphate buffer (0.1 M, pH 7.5), 100μl enzyme solution was placed in a new tube, 50 μl of 0.075 M ATChI and 50 μl of 0.01 M DTNB. The reaction began with the addition of the substrate (ATChI) and the reagent (DTNB). The change in absorbance at 412 nm was recorded for 5 min at a UV/Vis spectrophotometer (V-530).

α-and β-Esterase Assay:
Esterase activity was determined according to Van Asperen (1962) with some modification (Cao et al., 2008). Insects from each treatment were homogenized in phosphate buffer (0.1 M, pH 7.0). The homogenates were centrifuged at 12000 g for 15 min at 4 °C. The supernatants were transferred to new Eppendorf tubes for the assay. A 50 μl enzyme solution was incubated with 50 μl of α or ß-NA (30 mM) for 15 min at 30 °C. The reaction was stopped by adding 50 μl of stop solution (two parts of 1% Fast Blue RR and five parts of 5% sodium dodecyl sulfate). The absorbance was measured at 600 nm for the hydrolysis of α-NA and 550 nm for ß-NA at UV/Vis Spectrophotometer (V-530). Mean levels of total esterase activity cited were based on α and ßnaphthol standard curves at a UV/Vis spectrophotometer (V-530).

Glutathione S-Transferase:
The glutathione-S-transferase activity was measured as indicated by Habing et al. (1974). The larvae were homogenized in ice-cold phosphate buffer (0.1 M, pH 6.5). The homogenates were centrifuged at 12000 g for 15 min at 4 °C and the supernatants were transferred and kept at -40 °C for the assay. The reaction solution contained 100 μl supernatant, 10 μl CDNB (30 mM), and 10 μl GSH (50 mM). Enzyme activity was determined by continuous monitoring of the change in absorbance at 340nm for 3 min at 25 °C at a UV/Vis spectrophotometer (V-530).

Seeds Germination:
Germination tests were carried out according to International Standard Methods (ISM) (Anonymus, 1966) to find out the effect of the chemical and botanical seed protectants application on the germination percentage of treated faba bean.
Data analyses were expressed as mean ± standard error by using one-way ANOVA, through the SPSS computer program, statistically significant at p < 0.05 with least significant difference (LSD)

RESULTS
Based on the LC50, the susceptibility of C. maculatus adults varied among tested compounds (Table 1). Thiamethoxam showed the highest toxicity followed by deltamethrin which has moderate toxicity while the least effect was recorded with fennel seeds powder with LC50 value of 21.05, 85.1 and 36325.8 ppm, respectively. The numbers of eggs per female C. maculatus on faba bean seeds treated with LC10, LC25, LC50 and LC75 of deltamethrin, thiamethoxam and fennel seeds powder are given in table (2). The application of thiamethoxam significantly affected eggs laying at all concentrations used 1.43, 0.7, 0.4, and 0.3 for LC10, LC25, LC50 and LC75, respectively. While deltamethrin reduced the number of eggs laid counts in comparison with the control, but its effect was significant only for the high concentration tested LC50 (6.6 ppm) with complete banning of eggs laying at LC75. In contrast, fennel seeds powder failed to turn down the number of eggs laid by C. maculatus females compared with the control. The impact of tested powders on the fecundity was clear and there was a decrease in the number of eggs laid by females as powders concentration increased compared with the control.
The suppression of progeny production is as important a parameter as adult mortality to minimize grain damage by insect pests. During current studies, the insecticide application significantly affected progeny production. Adult emergence was significantly lower at all concentrations used of deltamethrin, thiamethoxam, and fennel seeds powders than the control and decreased as powders concentration amount increased. Moreover, progeny production was significantly suppressed at the rate of LC75 of both deltamethrin and thiamethoxam powders.
The residual activities of the contact treatment of faba bean seeds with LC95 of the deltamethrin, thiamethoxam and fennel seeds powders on C. maculatus adult's mortality (Table 3 and Fig. 1). Different mortality rates were noted among treatments at each exposure interval from the initial (24 h) to 14 months for both deltamethrin and thiamethoxam and 3 months in the case of fennel seeds. The percentage mortality of C. maculates adults after exposure for different periods on faba bean seeds treated with LC95 of deltamethrin and thiamethoxam provided the most efficient way in preserving seeds especially in the long run, complete protection to treated faba bean seeds with maintained after 24 h till 7 and 4 months for deltamethrin and thiamethoxam, respectively. A Gradual decrease in their toxicity occurred to reach 62.7 and 22.2% after 14 months post-treatment, for deltamethrin and thiamethoxam, respectively. In contrast, fennel seeds powder achieved high mortality of 95.3% only after 24 h, and then the effectiveness sharply declined after that period and caused the least mortality of 12% at 3 months and thereafter failed to produce any mortality against this insect species. * The mean difference is significant related to control followed by LSD (P<0.05). Table 3: Percentage mortality of C. maculatus adults exposed to faba bean seeds treated with LC95 of deltamethrin, thiamethoxam and fennel seeds powders after different periods (moths) Fig.1: Residual toxicity to Callosobruchus maculatus adults exposed to faba bean seeds treated with LC95 of deltamethrin, thiamethoxam and fennel seeds powders after different periods (moths) Data in table (4) indicated that the activity of AChE was significantly lower in all treatments related to the control associated with a much lower sublethal concentration of thiamethoxam (LC10). On the other hand, the activity of α esterase observed a significant decrease in all sublethal concentrations of thiamethoxam and deltamethrin. While the activity of ß esterase showed a significant decrease at LC10 of thiamethoxam only and all sublethal concentrations of deltamethrin compared with control. The GST activity exhibited a much lower value at LC10 sublethal concentration of thiamethoxam and a significant decrease at the other treatments. Table 4: Enzymes activities at adults of Callosobruchus maculatus exposed to sublethal concentrations of deltamethrin, thiamethoxam and fennel.
Enzyme activity is showed as mean ± SE followed by significant different by LSD (P<0.05).
The germination percentages of treated faba bean seeds with tested powders concerning storage period were presented (Table 5 and Fig. 2). At the initial application, germination percentages of faba bean seeds treated with deltamethrin, thiamethoxam, and fennel seeds powders at LC95 values and untreated seeds (control) were observed. The germination percentages within the seed protectants at the initial time were 96, 95 and 94% for deltamethrin, thiamethoxam and fennel seeds powders, respectively compared with 98% for control. While after 14 months of storage, the germination percentages of faba bean seeds treated with deltamethrin, thiamethoxam and fennel seeds powders were 84, 85 and 85%, respectively compared with 86% for control.

DISCUSSION
Based on the LC50, thiamethoxam proved the highest activity followed by deltamethrin, then finally F. vulgare seed powder (Table, 1). Previous findings confirmed thiamethoxam superiority against several species of stored product pests. For instance, Al-Attar (2016) reported that thiamethoxam was more toxic than three pyrethroid insecticides against five coleopterans stored products pest's species. In addition, Plodia interpunctella larvae were susceptible when exposed to maize treated with 50 ppm of thiamethoxam (Yue et al., 2003). Similarly, Rhyzopertha dominica and Sitophilus oryzae exposed to 1-4 ppm record 90-100% control (Fang et al. 2002). Also, thiamethoxam was the most effective against Tribolium confusum young larvae (Saglam et al., 2013). As thiamethoxam could be used with success against a wider range of species as a grain protectant (Rumbos et al., 2018).
The effect of tested powders on fertility was clear; all tested powders have a concentration-dependent effect on both egg-laying and adult emergence. Egg-laying status was a combination of some factors throughout the larval period treatments, but not because of adult treatments, only when adult females killed because of insecticide treatment, the number of laid eggs will be reduced. Similar findings with different grain protectants confirmed this notice. Regarding thiamethoxam, it provided high levels of parental mortality and progeny production suppression against examined stored-product beetle species (Rumbos et al., 2018). Similarly, thiamethoxam application at a low-level suppressed progeny production of Oryzaephilus surinamensis, Rhyzopertha dominica, Tribolium castaneum and Sitophilus zeamais on wheat and maize (Arthur et al., 2004). With deltamethrin, Stathers (2002) reported that deltamethrin prevented progeny emergence during the 8-week period. Our results confirmed several reports from multicountry which showed that deltamethrin was very effective against cowpea beetle (ZeroFly, 2015). Progeny production is a major function of any control measure, as it is crucial for long-term stored-products protection (Wakil et al., 2013). In our assay, C. maculatus was found to be more susceptible to thiamethoxam and had the highest value for F1 progeny reduction and did not respond to fennel seeds powders as compared to other insecticides had the lowest value for percentage F1 progeny reduction. Ruman et al. (2013) stated that F. vulgare plant powder was the lowest effective powder in the average of eggs/female, emergency of adult insect numbers with averages reached 90.60 eggs/female, 66.60 insect/female compared to Nigella sativa and Cyperus rotundus powders.
The residual effectiveness is a very important consideration when insect management tactics are to be designed for stored products. Our results indicate that deltamethrin, thiamethoxam and fennel seeds powders can be applied successfully against C. maculatus adults to protect seeds from infestation during storage. Regarding stability, fennel seeds powder is less stable than deltamethrin and thiamethoxam powders. Loss of biological activity of fennel seeds powder was probably due to the quick degradation of its active components (volatile oil). Our findings in line with Heydarzade and Moravvej (2012) who reported that the biological activity of F. vulgare oil was lost in a relatively short time and did not exceed 30 h. Ngamo et al. (2007) showed that the persistence of the biological activity of essential oils extracted from Hyptis spicigera and Lippia rugosa L. towards four stored-product pests, including C. maculatus, did not exceed 24 h. The decline in the efficacy of deltamethrin and thiamethoxam insecticides occurs slowly compared with fennel seeds powder. These results support the findings of Rai et al. (1987) that jute bags and jute cloth made cover impregnated with deltamethrin revealed good protection values 5 months after treatment against R. dominica and T. castaneum. Mishra and Pandey (2014) also found deltamethrin 2.5 WP at 40 mg/Kg wheat most effective of 8 treatments against S. oryzae after 3 months of storage. Singh et al. (1998), Pathak et al. (2002 found deltamethrin effective when wheat was dusted at 3 ppm. A similar result was reported by Pathak and Jha (2001) that deltamethrin was the most effective of all treatments (chlorpyrifos methyl, etrimfos, and malathion) after 180 days of storage on treated wheat.
Acetylcholinesterase plays a major role in the insect nervous system, which terminates nerve impulses by catalyzing the hydrolysis of the acetylcholine neurotransmitter. Several studies have established that pyrethroids can produce secondary effects on acetylcholinesterase (AChE) activity (Hossain et al., 2004) and has been reported that neonicotinoids reduce the AChE activity (Azevedo-Pereira et al., 2011;Badawy et al., 2015). The AChE inhibition one of the essential oils and monoterpene fumigation modes of action against stored grain insects (Kiran and Prakash, 2015). In the current paper, AChE exhibited a significant decrease at C. maculatus which treated with thiamethoxam, deltamethrin and fennel seed powders that agreed with Abdelsalam et al. (2020) who found that imidacloprid reduced acetylcholinesterase activity of the red palm weevil Rhynchophorus ferrugineus related to the dose (10, 15, 20 and 30 ppm). The significant inhibition of AChE was estimated in C. maculates treated with cassia oil and exhibited strong inhibition when treated with Guiera senegalensis galls methanol extract (El-Sayed et al., 2015;Sombié et al., 2018).
The disturbance could reflect protective physiological responses such as the increment of carboxylesterases (CarE), and glutathione S-transferases (GST) that play important roles in insecticide metabolism (Yu, 2004). Esterase and GST are a group of enzymes made up of protein (85%) and they play a critical role in the detoxification of toxic compounds that enter and exit from the insect body (Tarigan, and Harahap, 2016). Esterases main roles are degradation and hydrolysis ester bond found on pyrethroids and other pesticides, esterases act as regulatory enzymes and have been reported in all organisms for degradation functions (Bhatt et al., 2020). GST activity has been detected in all insects and in all larval and pupal stages (Hemmingway, 2000). Insect GSTs play an important role in protecting tissues from oxidative stress (Le et al., 2002). GST activity is used as an indicator of insecticide stress and examined in many species (Hou et al., 2008) and can be induced or inhibited as a response to toxicants exposure (Kolawole et al., 2011).
In this study, all sublethal treatments exhibited significantly decreased esterases and GST. The results were similar to the study conducted by Tarigan and Harahap (2016) who found that cardamom, cinnamon and nutmeg essential oils inhibit GST activity and reduced esterase activity on C. maculatus (concentration-dependent).
The results showed no adverse effect on the germination of faba bean seeds, due to the chemical and botanical seed protectants application. In all the treatments, seeds germination declined progressively with an increase in the storage period. The seeds treated with tested powders-maintained germination percent above the Minimum Seed Certification Standards (>75 %) up to the 12 weeks of storage period. The reduction in seed viability might be due to, temperature changes followed by changes in the moisture content of stored seeds during the storage periods (14 months). The present results were similar to those previously reported by El-Emam et al. (2010) who stated that prolonging the storage period over 12 months affected adversely seed germination. These results were also supported by Castellanos et al. (2017), who stated that a greater germination percentage of seeds treated with thiamethoxam doses between 200 and 300 mL 100 kg -1 of seeds, while doses below or above this range led to germination percentages similar to the control. This result was also supported by Almeida et al. (2011);Clavijo (2008); Almeida et al. (2009);Lauxen et al. (2010) who observed a similar response with the use of thiamethoxam in different seeds. In consistent with these results, Yadav et al. (2018) found that germination percent was found to be 84.33 and 83.00% when the seed was treated with emamectin benzoate 5SG at 40.0 g kg -1 seed and deltamethrin at 0.04 ml kg -1 seed respectively up to 9 months of storage.

Conclusions
The current study exhibited that sublethal concentrations of thiamethoxam, deltamethrin had potentially adverse effects and reduced the population growth on C. maculatus. Thiamethoxam may a promising insecticide and more effective to control C. maculatus and didn't affect seeds germination. Ethics approval and consent to participate: The manuscript does not contain any studies involving human participants, human data, or human tissue. Availability of data and materials: All data generated during this study are included in this published article. Competing interests: The authors declare that they have no competing interests. Funding: There was no funding for this work. Authors' contributions: AOA contributed to the design of the study and to conduct the biological part of the research and analyzing the biological obtained data, EAF designed the experiment and estimated the enzyme activity and analyzing the obtained data. The two authors cooperated in write and read the final manuscript and approved it.