Toxicity and biochemical effects of four pesticides on honey bee, Apis mellifera under laboratory conditions

Toxicidade e efeitos bioquímicos de quatro pesticidas na abelha Apis mellifera em condições de laboratório

Abstract

The widespread use of insecticides can cause negative side effects on pollinators, resulting undesirable effects in crop productivity. So, this study was conducted to evaluate the toxicity and biochemical changes in honey bees, Apis mellifera (Hymenoptera: Apidae) enzymes, treated with four insecticides i.e. lambda-cyhalothrin, hexythiazox, fenpyroximate and thiamethoxam under laboratory conditions. The obtained results revealed that thiamethoxam was extremely toxic to A. mellifera adults (LC₅₀=0.006 ppm) followed by lambda-cyhalothrin (LC₅₀=0.053 ppm) and fenpyroximate (LC₅₀=2.29 ppm) after 24 h of treatment, however, hexythiazox was relatively less toxic to bees (110.09 ppm). The sub-lethal concentration LC₂₅ and LC₅₀ of tested pesticides, reduced the activity of AChE, GST, MFO and esterase’s activities in honey bee adults, where, lambda-cyhalothrin and thiamethoxam induced the highest effects compared with other two pesticides and control. In contrast, the tested insecticides activated PPO enzyme at LC₂₅ concentration, while PPO activity was reduced after treating adult bees with LC₅₀. Lambda-cyhalothrin, thiamethoxam, fenpyroximate and hexythiazox disrupted the physiology of honey bees, thereby reducing the efficiency of this beneficial pollinator. Overall, the obtained results are valuable not only in evaluating the toxicity of common insecticides onto honey bees, but also in highlighting the validity of enzyme activities as appropriate indicators for exposure to agrochemicals.

Keywords:
Apis mellifera; toxicity; detoxification enzyme; side effects; biochemical analysis

Resumo

O uso generalizado de inseticidas pode causar efeitos colaterais negativos nos polinizadores, o que gera um impacto indesejável na produtividade agrícola. Assim, o presente estudo foi realizado com o objetivo de avaliar a toxicidade e as alterações bioquímicas em abelhas melíferas, utilizando enzimas da Apis mellifera (Hymenoptera: Apidae) tratadas com quatro inseticidas, especificamente o lambda-cialotrina, hexitiazox, fenpiroximato e o tiametoxam, em condições de laboratório. Após 24 h de tratamento, os resultados obtidos revelaram que o tiametoxam foi extremamente tóxico para A. mellifera adultos (CL₅₀=0,006 ppm), seguido do lambda-cialotrina (CL₅₀=0,053 ppm) e o fenpiroximato (CL₅₀=2,29 ppm), sendo o hexitiazox relativamente menos tóxico para as abelhas (110,09 ppm). As concentrações subletais LC₂₅ e LC₅₀ dos pesticidas testados reduziram a atividade de AChE, GST, MFO e atividades de esterase em abelhas adultas, com o lambda-cialotrina e o tiametoxam induzindo efeitos mais expressivos em comparação com os outros dois pesticidas e o controle. Em contraste, os inseticidas testados ativaram a enzima PPO na concentração LC₂₅, enquanto a atividade da PPO foi reduzida após o tratamento de abelhas adultas com LC₅₀. O uso de lambda-cialotrina, tiametoxame, fenpiroximato e hexitiazox perturbou a fisiologia das abelhas melíferas, reduzindo assim a eficiência deste polinizador benéfico. No geral, os resultados aqui obtidos são valiosos não apenas para avaliar a toxicidade dos inseticidas comuns nas abelhas melíferas, mas também para destacar a validade das atividades enzimáticas como indicadores apropriados para a exposição aos agroquímicos.

Palavras-chave:
Apis mellifera; toxicidade; enzima de desintoxicação; efeitos colaterais; análise bioquímica

1. Introduction

Honey bees, Apis mellifera had great importance in agricultural fields, as a pollinator for many major crops and wild plants around the world, and considered one of the important components for biodiversity and food security (Colin et al., 2004). Also, in Africa, A. mellifera contributes plays an important role in pollinating most of agricultural crops (van Engelsdorp and Meixner, 2010; Khalifa et al., 2021). Honey bees have great values in the production of healthy food such as honey, wax, pollen, propolis, royal jelly and venom also, it play a major role in increasing income and medical products (Ratnakar et al., 2017; Hung et al., 2018). Generally, insect pollination is required to increase agricultural products (Calderone, 2012; Jordan et al., 2021).

Insect pests cause significant damage and a threat to the productivity of many field crops. To minimize economic losses, producers have used many control methods, the most common of which are insecticides (Oerke, 2005; Aktar et al., 2009; Guedes et al., 2016). Worldwide, the use of insecticides in crops is considered as one of the main reasons responsible for decline in bee population (Pires et al., 2016). In addition Hristov et al. (2021) reported that the main potential cause of colony collapse is interaction between environmental stress factors, particularly exposure to pesticides and pathogens, so, it is difficult to detect the main reasons for loss of colonies. Also, there are some factors negatively affect honey bee colonies, as bee-keeping practices, pests, diseases, pesticide use, agricultural practices, and climate changes.

Iwasa et al. (2004) and Laurino et al. (2011) reported that neonicotinoids are extensively used worldwide for the control of major agricultural crop pests in the field such as aphids, leafhoppers, and whiteflies demonstrated high toxic effect to A. mellifera. Also, Goulson et al. (2015) found that, although the use of neonicotinoid pesticides has achieved high efficiency in controlling pests, it cause harm to non-target organisms such as pollinating bees. Moreover, Castilhos et al. (2019) reported that pesticide exposure induced colony losses, and found that the insecticides such as Imidacloprid, Thiamethoxam, and Clothianidin are highly toxic to bees. In addition, Serra et al. (2021) reported that the most common ways for exposing honey bees to pesticides are by contact as well as the residues on plants and ingesting contaminated food, caused histological alterations in bee midgut. Moreover, Dai et al. (2018), Fine et al. (2017) and Osborne (2012) reported that bee workers were highly contact with pesticide residues during foraging for food source. Also, Zhao et al. (2022) cleared that the uncritical use of neonicotinoids, pyrethroids, chlorantraniliprole, spinosad, flupyradifurone and sulfoxaflor insecticides led to harmful effects on honey bee growth and development, in addition to decrease their foraging activity and pollination services. Recently, Souza et al. (2024) found that exposure to thiamethoxam in the field led to a minimal impact on population growth or mortality of bees in the colonies, besides that, thiamethoxam caused severe toxicity to immature honeybees inside the hives.

With respect to the effect of some environmental pollutants such as pesticides on honey bee enzymes as a biomarkers to indicate any behavioral and functional changes in both target and non-target insects exposed to high or sublethal doses of insecticides, Acetyl cholinesterase (AChE), carboxylesterase (CarEs), glutathione S-transferase (GST), and polyphenol oxidase (PPO) are considered examples of these enzymes that can be assayed. Tu et al. (2009), reported that AChE has been widely used as a biomarker of general exposure to pollutants, especially organophosphate and carbamate pesticides. However, the activities of other enzymes such as Glutathione S-transferases and carboxylesterase involved in the detoxification and removal of a wide variety of toxic compounds (Hinton et al., 1995). In addition, the biochemical and physiological effects of insecticides can render individual honeybees unable to make their duty easily, thus affecting the colony performance (Chakrabarti et al., 2020). Glutathione S-transferases and Mixed-function oxidase are considered important enzymes and are responsible for insecticide detoxication on insects (Bird et al., 2022). Also, Zhu et al. (2017) found that the activity of Lambda-cyhalothrin on GST enzyme was increased while the toxicity was increased. Roudsari et al. (2022) found that the sub-lethal concentration of imidacloprid was reduced the activity of acetylcholinesterase (AChE) and glutathione S-transferase (GST). Furthermore, exposed honey bee larvae to imidacloprid (0.16 mg a.i./l) increased the mortality of the treated group compared to untreated controls, but this difference was not statistically significant, and concluded that imidacloprid disrupt the physiology of honey bees, thereby reducing the efficiency of this beneficial pollinator. Recently, Choi et al. (2024) pointed that Lambda-Cyhalothrin may affect the development of honey bees and induced significant changes in detoxification (GST), antioxidative (SOD and CAT), and AChE in developmental stages of honey bees.

From the aforementioned background, and the previous studies, it became clear that thiamethoxam induced a high risk to honey bees. In addition, in Egypt, thiamethoxam is used in controlling many insects, and also applied as crop spraying and seed dressing for protection from soil insects. Therefore, in this study, this pesticide was used in comparison with three other pesticides belonging to different groups in terms of their toxicity to honey bees in the laboratory after different time periods. Moreover, to study the effects of sublethal effects on a group of enzymes as biomarkers within bees in order to find out the safest pesticide on bees in preparation for recommendation to use in integrated pest control programs.

2. Materials and Methods

2.1. Insecticides and chemicals

Four pesticides with different mode of actions were selected for evaluation on honey bee, Apis mellifera L. (Hymenoptera: Apidae) adults. Further details about the recommended dose rates, group, trade name, and mode of action are provided in Table 1.

Table 1
List of tested pesticides including group, mode of action and rate of use.

Common name	Trade name	Group & Mode of action	used rates
Lambda-cyhalothrin	Lambda® 5% EC	Pyrethroids& Disrupt the normal functioning of the nervous system in an organism	20 mL/100 L water
Hexythiazox	Magnifico 5% EC	Thiazolidionone& Non-systemic acaricide with contact and stomach action. Good translaminar activity. Has ovicidal, larvicidal, and nymphicidal activity.	20 mL/100 L water
Fenpyroximate	Ortus 5% SC	Pyrazole & a mitochondrial NADH: ubiquinone reductase inhibitor	50 mL/100 L water
Thiamethoxam	Actara 25% WG	Neonicotinoid& Disrupting the nervous system of targeted pest either ingests or absorbs the poison into its body.	20 g/100 L water

2.2. Honey bee samples

The foraging young worker bees Apis mellifera L. (Hymenoptera: Apidae) (3-7 days after emergence) used in this experiment were obtained from the apiary of the Economic Entomology Department of the Faculty of Agriculture, Menoufia University, and transported to the laboratory in plastic boxes covered with screen, kept at 25 ± 2 °C and 65 ± 5% relative humidity and they were fed on 50% sugar cane solution until used.

2.3. Laboratory experiments

2.3.1. Toxicity of tested insecticides on adult workers of honey bee

Foraging young adult worker bees (3–7 days old) were exposed to oral toxicity test, where five concentrations of each insecticide were prepared in 50% (w/v) sucrose. Prior to treatment with insecticide, young adult worker bees (10/jar) were starved for 24 hr and anesthetized by exposure to carbon dioxide gas for 1 min. A piece of cotton was saturated with 5 mL of each concentration and attached to the upper side of the jar, and then the jars were covered with a piece of muslin. Bees were left to feed on a single dose of each tested insecticide for 24 h, then cotton was removed and replaced with other one contain only 50% sugar solution and replaced with another one daily until the end of the test. Control was fed with 50% sugar cane solution and incubating bees at 25 ± 2 °C and 65 ± 5% relative humidity, 12:12 (L:D) photoperiod. All treatments were replicated three times. The bees were considered dead if unable to walk when prodded with a fine hair brush. Mortality percentages were recorded 24,48 and 72 days of treatment and corrected according to Abbott formula (1925) then analyzed by probit analysis to calculate the lethal concentrations (LC₅₀ and LC₉₀) with their corresponding fiducial limits at 95% (FL).

2.3.2. Detoxification enzyme assays

Specific activities of AcetylCholinEsterase(AChE), Esterase and Polyphenol oxidase enzymes were determined in the head of the surviving honey bee workers treated with LC₂₅ and LC₅₀ of each pesticide after 12, 24 and hrs of oral treatment. Surviving bees were anesthetized as mentioned above and the head was dissected and rinsed in ice-cooled phosphate buffer (pH 7.0). In addition to the three enzymes stated above, Glutathione S-transferase (GST) and Mixed Function Oxidase MFO were extracted from the midgut of tested bees.

To measure the activity of the aforementioned enzymes, crude extract of their assigned tissues was used. The extraction solution consisted of 10 mM NaCl, 1% (w/v) Triton X-100, and 40 mM sodium phosphate buffer (pH 7.4). The tissues were homogenized in the extraction solution using a glass/ Teflon homogenizer on ice. The homogenate was filtered using cheese cloth and then centrifuged at 10,000 rpm for 20 min at 4 °C. The tissue pellet was subjected to extraction and centrifugation three times with phosphate buffer (pH 7.4). The three recovered supernatant fractions containing crude enzyme were mixed and used immediately for assaying AChE, MFO, PPO, Esterase and GST activities or stored at −20 °C until determination. All procedures were carried out at 4 °C, and all of the experiments were performed in triplicate.

2.3.3. Esterase activity

The esterase activity was determined using α-Na and β-Na as substrates according to van Asperen (1962) with slight modification. The total reaction consisting of 20 μL of enzyme solution, 200 μL of the substrate (30 mM in acetone),and 50 μL of phosphate buffer (0.1 M, pH 7.5) was incubated for 20 min followed by the addition of 50 μL fast blue RR (1 mg/mL in buffer). Optical density was read at 450 and 540 nm forα-Na and β-Na respectively, every 2 min for 20 min using a microplate reader (ELX808 Bio-Tek).

2.3.4. Glutathione S-transferases (GST) activity

The CDNB was used as substrate to measure the GST activity according to the method of Habig et al. (1974) with slight modification. Ten microliter of enzyme extract was mixed with 200 μL of 63 mM CDNB and 2.5 mM reduced glutathione (GSH) in sodium phosphate buffer (0.1 M, pH 6.5). Absorbance was recorded using the microplate reader continuously every 30 s for 20 min at 340 nm.

2.3.5. AcetylCholinEsterase (AChE) activity

AChE activity was determined using ATChI as the substrate according the method of Ellman et al. (1961) with slight modification. Briefly, the reaction mixture consisting of ATChI (1.5 mM), DTNB (1 mM), and an enzyme preparation (40 μL) was prepared in a final volume of 240 μL with phosphate buffer (0.1 M, pH 7.5). The absorbance was measured continuously every 2 min for 30 min at 415 nm. Enzyme activity was calculated using the extinction coefficient of 0.136 mM−1 cm−1 for 5-thio-2-nitrobenzoate.

2.3.6. Mixed-function oxidases (MFO) activity

Mixed-Function Oxidases (MFO) activity was tested according to Hansen and Hodgson (1971) and Moustafa et al. (2021). First, 100 μL of 2mM p-nitro anisole was incubated with 90 μL of homogenate sample for 2 min at 27 °C, and 10 μL of 9.6mM NADPH was added to initiate the reaction. Then, the activity of MFO was measured at 405 nm for 15 min using molecular devices of microplate reader (Clindiag-MR-96, ISO09001:2008, Steenberg, Belgium). Finally, the standard curve of p-nitrophenol was used to calculate the MFO activity.

2.3.7. Poly phenol oxidase (PPO) enzyme activity

The activity of PPO enzyme was measured, using 100 μL of the crude enzyme, 700 μL of 0.2 M pyrocatechol substrate, and 700 μL of 0.1 M phosphate buffer (pH 6.8) were mixed and incubated for 5 min at 30 °C. The absorbance of developed color due to the oxidation of pyrocatechol (Sigma-Aldrich, St. Louis, MO, USA) by PPO was monitored at 420 nm as described by Fattouch et al. (2010). One unit (U) of toxicity of each insecticide on honey bees worker PPO activity was defined as the amount of enzyme that caused an increase in color absorbance by 0.001 optical densities per min. The specific activity of PPO was defined as U/mg protein.

2.4. Statistical analysis

LC₅₀ and LC₂₅ were determined using probit analysis with the software Polo-Plus (LeOra Software, 2007). Statistical analysis was performed by comparisons of the means using T-test in SPSS software (version 22) (P-value < 0.05).

3. Results

3.1. Toxicity of lambda-cyhalothrin, hexythiazox, fenpyroximate, and thiamethoxam on honey bee foragers, A. mellifera L.

Toxicity bioassays of formulated lambda-cyhalothrin, hexythiazox, fenpyroximate, and thiamethoxam on honey bee workers, A. mellifera L. are summarized in Table 2. The obtained data show that thiamethoxam and lambda-cyhalothrin were obviously the most toxic tested pesticide on A. mellifera honey bee foragers (LC₅₀ = 0.006 and 0.053 ppm) among the four tested insecticides, while hexythiazox was the lowest toxic pesticide against the foragers (LC₅₀ = 110.09 ppm) and fenpyroximate was relatively low toxicity to bees (LC₅₀ = 2.29 ppm) after 24 h of oral exposure.

Table 2
Acute toxicity of Lambda-cyhalothrin, Hexythiazox, Fenpyroximate, and Thiamethoxam against honey bees (A. mellifera L.) by oral application after 24 hr of treatment.

Pesticides	LC50 (ppm) (CL)	Slope ±SE	Toxicity index (LC50)	LC90 (ppm) (CL)	Toxicity index (LC90)
Lambda-cyhalothrin	0.053 (0.0002-0.38)	0.402±0.11	26.2	81.45 (18.5-2298.88)	21.95
Hexythiazox	110.09 (48.12-430.89)	0.702±0.15	0.005	7328.99 (2263.8-487481.09)	0.24
Fenpyroximate	2.29 (1.54-17.05)	0.76±0.11	26.2	157.75 (54.53-850.7)	11.1
Thiamethoxam	0.006 (0.0-0.10)	0.37±0.12	100	17.53 (3.25-590.08)	100

• Toxicity index = [(LC₅₀ or LC₉₀ of the efficient pesticide/LC₅₀ or LC₉₀ of the efficient pesticide) x 100]. CL = Confidence limits.

As for the toxicity after 48 h of exposure Table 3, the obtained data revealed that LC₅₀ values were decreased in all tested insecticides. Lambda-cyhalothrin and thiamethoxam were obviously the most harmful to honey bees (LC₅₀ = 0.001 and0.002 ppm), while hexythiazox showed the lowest harm to the foragers (LC₅₀ = 16.735 ppm) and fenpyroximate was relatively low toxicity to bees (LC₅₀ = 1.49 ppm) after 48 h of oral exposure.

Table 3
Acute toxicity of Lambda-cyhalothrin, Hexythiazox, Fenpyroximate, and Thiamethoxam against honey bees (A. mellifera L.) by oral application after 48 hr of treatment.

Pesticides	LC50(ppm) (CL)	Slope ±SE	Toxicity index (LC50)	LC90(ppm) (CL)	Toxicity index (LC90)
Lambda-cyhalothrin	0.001 (-)	0.311±0.13	100	18.74 (-)	60.4
Hexythiazox	16.735 (6.87-41.13)	0.62±0.11	50.2	1934.95 (432.69-36458.52)	0.6
Fenpyroximate	1.49 (0.57-3.51)	0.62±0.09	50.2	172.17 (50.13-1335.64)	10.9
Thiamethoxam	0.002 (-)	0.34±0.12	91.2	11.31 (-)	100

• Toxicity index = [(LC₅₀ or LC₉₀ of the efficient pesticide/LC₅₀ or LC₉₀ of the efficient pesticide) x 100]. CL = Confidence limits.

The obtained data show that, the toxicity of all tested insecticides Table 4 were very low, where LC₅₀ was decreased to 0.0001, 0.0004, 0.09 and 3.12 ppm for lambda-cyhalothrin, thiamethoxam, fenpyroximate and hexythiazox, respectively after 72 h of exposure.

Table 4
Acute toxicity of Lambda-cyhalothrin, Hexythiazox, Fenpyroximate, and Thiamethoxam against honey bees (A. mellifera L.) by oral application after 72 hr of treatment.

Pesticides	LC50 (ppm) (CL)	Slope ±SE	Toxicity index (LC50)	LC90 (ppm) (CL)	Toxicity index (LC90)
Lambda cyhalothrin	0.0001 (-)	0.251±0.12	100	13.58 (-)	100
Hexythiazox	3.12 (1.04-7.31)	0.64±0.12	0.003	314.43 (91.19-3230.24)	4.3
Fenpyroximate	0.09 (0.003-0.45)	0.37±0.1	0.11	277.41 (31.85-89884.41)	4.9
Thiamethoxam	0.0004 (-)	0.28±0.12	25	13.69 (-)	99.2

• Toxicity index = [(LC₅₀ or LC₉₀ of the efficient pesticide/LC₅₀ or LC₉₀ of the efficient pesticide) x 100]. CL = Confidence limits.

3.2. Effects of tested insecticides on acetyl cholinesterase (AChE) activity

The data in Table 5 revealed that the activity of AchE was decreased in all tested pesticides after treated with LC₂₅ after 12, 24 and 48 h of exposure and there were significant differences in activity of AchE between all treatments and control. Where, thiamethoxam induced the highest decrease in the enzyme activity (1.21, 1.18 and1.11 nmoles ATCh I hydrolyzed/mg protein/min) followed by lambda-cyhalothrin (1.51, 1.43 and nmoles ATCh I hydrolyzed/mg protein/min) after 12, 24 and 48 h of treatment, respectively.

Table 5
AchE activity in honey bees (A. mellifera L.) adults after treated with LC₂₅ and LC₅₀ of tested pesticides.

treatments	Specific activity (nmoles ATCh I hydrolyzed/mg protein/min)			Mean of AchE activity (nmoles ATCh I hydrolyzed/mg protein/min)
	After treated with LC25
	12 h.	24h.	48 h.
Lambda-cyhalothrin	1.51 bc±0.05	1.43 d±0.09	1.41 d ±0.09	1.45 d±0.09
Hexythiazox	2.17 a±0.05	2.11 b±0.05	2.10 b±0.05	2.13 b±0.06
Fenpyroximate	1.73 b±0.09	1.72 c±0.09	1.65 c±0.09	1.7 c±0.1
Thiamethoxam	1.21 c±0.04	1.18 d±0.04	1.11 e±0.09	1.17 e±0.05
control	2.47 a±0.09	2.54 a±0.09	2.51 a±0.09	2.51 a±0.09
LSD (0.05)	0.31	0.27	0.23	0.23
F	25.72	39.18	57.90	53.53
P-value	0.0000	0.0000	0.0000	0.0000
	After treated with LC50
	12 h.	24h.	48 h.
Lambda-cyhalothrin	1.20 d±0.09	1.18 d±0.05	1.14 c±0.05	1.17 d±0.05
Hexythiazox	1.93 b±0.1	1.84 b±0.09	1.80 b±0.09	1.86 b±0.09
Fenpyroximate	1.51 c±0.09	1.46 c±0.09	1.41 c±0.09	1.46 c±0.09
Thiamethoxam	1.00 d±0.05	0.93 d±0.14	0.82 d±0.09	0.92 e±0.09
control	2.47 a0.09	2.54 a0.14	2.51 a±0.4	2.51 a±0.1
LSD(0.05)	0.27	0.26	0.32	0.22
F	47.44	58.93	42.48	83.17
P-value	0.0000	0.0000	0.0000	0.0000

• P = Probability; LSD = Least statistical difference. The same letters means no significant difference at 5% level. Each value is the mean of three samples ±SE.

Thiamethoxam recorded the lowest mean of enzyme activity (1.17 nmoles ATCh I hydrolyzed/mg protein/min) followed by lambda-cyhalothrin (1.45 nmoles ATCh I hydrolyzed/mg protein/min) after honey bees treated with LC₂₅ concentration.

AchE enzyme activity was significantly decreased after treated with LC₅₀ of all tested pesticides compared to control and that treated with LC₂₅ (Table 5). Thiamethoxam induced the highest decrease in AchE activity recording 1.00, 0.93 and 0.82 nmoles ATCh I hydrolyzed/mg protein/min after 12, 24 and 48 h of treatment with LC50 concentration, followed by lambda-cyhalothrin which recording enzyme activity 1.20, 1.18 and 1.14 nmoles ATCh I hydrolyzed/mg protein/min after 12, 24 and 48 h of treatment, respectively. Thiamethoxam recorded the lowest mean of AchE activity 0.92 nmoles ATCh I hydrolyzed/mg protein/min).

Generally, the AchE activity was decreased after treated with LC₂₅ and LC₅₀ of tested insectiides as well as it was decreased with the increasing of The enzyme activity decreased with insecticide concentrations.

3.3. Effects of tested insecticides on Glutathione S-Transferase activity

Obtained results in Table 6 represent the effect of lambda-cyhalothrin, hexythiazox, and fenpyroximate on GST activity in honey bee workers treated with LC₂₅ and LC₅₀ after 12, 48 and 48 h. Results revealed that enzyme activity were decreased after treated honey bee adults with LC₂₅ of thiamethoxam and lambda-cyhalothrin compared with other treatments and control recording enzyme activity 0.212, 0.231 and 0.210 μmole /bee/mg of protein and 0.242, 0.257 and 0.237 μmole/bee/mg of protein after 12, 24 and 48 h of treatments, respectively.

Table 6
Glutathione S-transferase (GST) activity in honey bees (A. mellifera L.) adults after treated with LC₂₅ and LC₅₀ of tested pesticides.

treatments	GST (μmole/bee/mg of protein)			Mean of GST (μmole/bee/mg of protein)
	After treated with LC25
	12 h.	24h.	48 h.
Lambda-cyhalothrin	0.242 b±0.01	0.257 c±0.01	0.237 b±0.0.01	0.306 b±0.06
Hexythiazox	0.410 a±0.09	0.453 a±0.01	0.447 a±0.06	0.437 a±0.01
Fenpyroximate	0.347a±0.05	0.368 b±0.02	0.361 a±0.01	0.359 ab±0.02
Thiamethoxam	0.212 b±0.005	0.231 c±0.01	0.210 b±0.02	0.218 c±0.006
control	0.421 a±0.01	0.462 a±0.02	0.436 a±0.006	0.44 a±0.1
LSD (0.05)	0.082	0.082	0.083±0.1	0.087
F	13.65	16.86	17.61	11.51
P-value	0.0005	0.0002	0.0002	0.0009
After treated with LC50
Lambda-cyhalothrin	0.621 b±0.01	0.646 b±0.02	0.612 a±0.06	0.626 b±0.01
Hexythiazox	0.426 c±0.01	0.463 c±0.01	0.441 c±0.05	0.443 c±0.02
Fenpyroximate	0.537 b±0.01	0.584 b±0.02	0.521 c±0.06	0.547 b±0.02
Thiamethoxam	0.862 a±0.06	0.841a±0.01	0.821 a±0.06	0.841 a±0.06
control	0.421 c±0.006	0.462 c±0.09	0.436 c±0.01	0.44 c±0.1
LSD (0.05)	0.09	0.097	0.088	0.091
F	40.87	25.77	32.93	33.11
P-value	0.0000	0.0000	0.0000	0.0000

• P = Probability; LSD = Least statistical difference. The same letters means no significant difference at 5% level. Each value is the mean of three samples ±SE.

Thiamethoxam and lambda-cyhalothrin recorded the lowest mean of Glutathione S-transferase activity 0.359 and 0.306 μmole/bee/mg of protein, compared with other treatments and control.

With respect to the effect of lambda-cyhalothrin, hexythiazox, fenpyroximate on GST activity in bee workers treated with LC₅₀ after 12, 48 and 72 h, data in Table 6 revealed that the enzyme activities were increased compared with that of LC₂₅ after 12, 24 and 48 h . The enzyme activities were increased in thiamethoxam, fenpyroximate and lambda-cyhalothrin treatments after 12 and 24 h of exposure recording 0.212 and 0.231 μmole/bee/mg of protein, 0.347 and 0.368 μmole/bee/mg of protein, and 0.242 and 0.257 μmole/bee/mg of protein, respectively. In contrast, there was no significant differences between hexythiazox and control after 12 and 24 h of treatment, whereas, there were significant differences in GST activity after treating bee workers with LC₅₀ of thiamethoxam and lambda-cyhalothrin recording 0.821 and 0.612 μmole/bee/mg, respectively, compared with control and other treatments recording the highest mean of GST activity as 0.841 and 0.626 μmole/bee/mg, respectively.

Generally, thiamethoxam and lambda-cyhalothrin revealed the lowest GST activity after treated honey bee workers with LC_25, as well as the highest enzyme activity was recorded after treating bee workers with LC_50, with positive relation between insecticides concentrations and enzyme activity, where the enzyme activity was increased by increasing insecticides concentrations.

3.4. Effects of tested insecticides on Mixed function oxidase activity

Data in Table 7 show the effect of lambda-cyhalothrin, hexythiazox, fenpyroximate and thiamethoxam on MFO activity in bee workers after 12, 24 and 48 h of exposure at the LC₂₅ and LC₅₀. MFO activity was significantly reduced after treating with LC₂₅ of the tested insecticides 12, 24 and 48 h of exposure except hexythiazox after 12 h. There were negative correlation between enzyme activity and time after treatment, where, MFO activity was decreased by increasing exposure time. Lambda-cyhalothrin induced the highest decrease in enzyme activity as 0.10 and 0.091 μmole/bee/mg of protein, after 24 and 48 h, respectively. Also, Lambda-cyhalothrin recorded the lowest mean of MFO 0.101 μmole/bee/mg of protein.

Table 7
Mixed-Function Oxidases (MFO) enzyme activity of honey bees (A. mellifera L.) adults after treated with LC₂₅ and LC₅₀ values of tested pesticides.

Treatments	MFO (μmole/bee/mg of protein)			Mean of MFO (μmole/bee/mg of protein)
	After treated with LC25
	12 h.	24h.	48 h.
Lambda-cyhalothrin	0.113 b±0.01	0.10 c±0.03	0.091 d±0.03	0.101 b±0.006
Hexythiazox	0.139ab±0.01	0.134 b±0.01	0.127 b±.006	0.133 b±0.0.01
Fenpyroximate	0.129 b±0.01	0.120 b±0.01	0.119 bc±0.006	0.123 b±0.06
Thiamethoxam	0.124 b±0.01	0.121 b±0.01	0.110 c±0.006	0.118 b±0.06
control	0.169 a±0.02	0.171 a±0.02	0.173 a±0.02	0.171 a±0.02
LSD(0.05)	0.03	0.014	0.014	0.034
F	4.86	34.46	45	5.67
P-value	0.020	0.0000	0.0000	0.012
After treated with LC50
Lambda-cyhalothrin	0.10 c±0.03	0.089 c±0.01	0.061 e±0.01	0.083 b±0.02
Hexythiazox	0.127 b±0.01	0.121 b±	0.119 b±0.06	0.102 b±0.001
Fenpyroximate	0.121 b±.06	0.10 c±0.06	0.10 c±0.03	0.099 b±0.02
Thiamethoxam	0.099 c±0.06	0.091 ±0.03	0.079 d±0.02	0.062 c±0.01
control	0.169 a±0.02	0.171 a±0.01	0.173 a±0.02	0.171 a±0.01
LSD (0.05)	0.018	0.018	0.018	0.018
F	23.78	34.65	59.8	49.23
P-value	0.0000	0.0000	0.0000	0.0000

• P = Probability; LSD = Least statistical difference. The same letters means no significant difference at 5% level. Each value is the mean of three samples ±SE.

The effect of lambda-cyhalothrin, hexythiazox, fenpyroximate and thiamethoxam on MFO activity of bee workers after 12, 24 and 48 h of exposure to LC₅₀ concentration are shown in Table 7. The obtained data clearly indicated that the enzyme activity was significantly decreased after 12, 24 and 48 h of treatment compared with LC₂₅ and control.

Lambda-cyhalothrin recorded 0.061 μmole/bee/mg of protein, and thiamethoxam recorded 0.079 μmole/bee/mg of protein) as the highest decrease in MFO enzyme after 48 h of treatment compared with other tested insecticides and control, moreover the lowest mean of MFO was recorded with thiamethoxam as 0.062 μmole/bee/mg of protein, compared with other tested insecticides and control.

It can be concluded that the MFO activity in adults of bees was strongly inhibited by lambda-cyhalothrin at LC₂₅ and LC₅₀ after 48 h . All tested insecticides, significantly reduced the amount of tested enzymes at LC₂₅ and LC₅₀ after 12, 24 and 48 h of treatment with negative correlation between enzyme activity and time after treatment, as well as insecticide concentrations.

3.5. Effects of tested insecticides on Polyphenol Oxidase (PPO) activity

Results in Table 8 represent the effect of lambda-cyhalothrin, hexythiazox, fenpyroximate and thiamethoxam on PPO in bee workers 12, 24 and 48 h of exposure to LC₂₅ and LC₅₀ concentrations. Results show that PPO enzyme was significantly activated at LC₂₅ treatment of tested insecticides after 12, 24 and 48 h of exposure compared to the untreated bees. Lambda-cyhalothrin recorded 3.40, 3.52, and 3.70 U/mg protein, and thiamethoxam recorded 3.31, 3.40 and 3.49 U/mg protein inducing the highest activation of enzyme activity after 12, 24 and 48 h of exposure, respectively.

Table 8
Poly Phenol Oxidase (PPO) enzyme activity of honey bees (A. mellifera L.) adults after treated with LC₂₅ and LC₅₀ values of tested pesticides.

treatments	(U/mg protein)			Mean of(PPO) enzyme activity (U/mg protein)
	After treated with LC25
	12 h.	24h.	48 h.
Lambda-cyhalothrin	3.40 a±0.2	3.52 a±0.2	3.70 a±0.2	3.54 a±0.2
Hexythiazox	2.89 b±0.2	2.99 b±0.1	3.12 d±0.07	3 ab±0.3
Fenpyroximate	2.97 b±0.2	3.10 b±0.06	3.31 c±0.1	3.13 ab±0.08
Thiamethoxam	3.31 a±0.2	3.40 a±0.2	3.49 b±0.2	3.4 a±0.2
control	2.15 c±0.09	2.19 c±0.1	2.32 e±0.01	2.22 b±0.06
LSD(0.05)	0.33	0.27	0.16	0.88
F	22.82	37.52	138.48	3.43
P-value	0.0001	0.000	0.0000	0.052
After treated with LC50
Lambda-cyhalothrin	1.53 b±0.2	1.42 b±0.1	1.31 b±0.06	1.42 b±0.1
Hexythiazox	2.37 a±0.1	2.50 a±0.2	2.45 a±0.1	2.44 a±0.2
Fenpyroximate	2.22 b±0.06	2.37 a±0.1	2.43 a±0.1	2.34 a±0.1
Thiamethoxam	1.76 b±0.2	1.71 b±0.2	1.63 b±0.2	1.7 b±0.2
control	2.15 a±0.09	2.19 a±0.06	2.32 a±0.1	2.22 a±0.07
LSD (0.05)	0.27	0.38	0.36	0.35
F	16.26	14.25	20.6	16.23
P-value	0.0002	0.0004	0.0001	0.0002

• The same letters means no significant difference at 5% level. Each value is the mean of three samples ±SE.

With respect to the effect of tested insecticides at LC₅₀ on PPO activity in bee workers, 12, 24 and 48 h of exposure, the obtained data revealed that the enzyme activity was significantly decreased after 12, 24 and 48 h of exposure compared with LC₂₅ treatments and control. Lambda-cyhalothrin recorded 1.53, 1.42, and 1.31 U/mg protein and thiamethoxam recorded 1.76, 1.71 and 1.63 U/mg protein, inducing the highest activation of enzyme activity after 12, 24 and 48 h of exposure, respectively. Also, lambda-cyhalothrin and thiamethoxam recorded the lowest mean of (PPO) enzyme activity as 1.42 and 1.7 U/mg protein, respectively.

Generally, there were negative correlation between insecticide concentration and enzyme activity, where, PPO activity was increased with low concentrations of tested insecticides, while it was decreased by increasing of insecticide concentrations s. Lambda-cyhalothrin and thiamethoxam induced the highest activation of enzyme activity in bee workers treated with LC₂₅, while, the two insecticides induced the highest decrease of enzyme activity in bee workers treated with LC₅₀ after 12, 24 and 48 h of treatment.

3.6. Effects of tested insecticides on Esterase enzyme activity

The effect of insecticides on the activity of EST in worker bees, oral treated with LC₂₅ and LC₅₀ after 12, 24 and 48 h by was determined in Table 9. It was obvious that the EST enzyme activity was significantly decreased 12 and 24 h of treating with LC₂₅ of lambda-cyhalothrin (0.051 and 0.04 μmol/min/mg protein) and thiamethoxam (0.061 and 0.050 μmol/min/mg protein), whereas, there were no significant differences between hexythiazox and fenpyroximate and control.Lambda-cyhalothrin, fenpyroximate and thiamethoxam significantly decreased enzyme activity after 48 h of treatment. Lambda-cyhalothrin and Thiamethoxam induced the highest mean of esterase activity as 0.037 and 0.068 μmol/min/mg proteins, respectively.

Table 9
Activity of Esterase enzyme (EST) of honey bees (A. mellifera L.) adults after treated with LC₂₅ and LC₅₀ values of tested pesticides.

treatments	Activity of Esterase (β- naphthyl acetate) (μmol/min/mg protein)			Mean of Esterase activity(μmol/min/mg protein)
	After treated with LC25
	12 h.	24h.	48 h.
Lambda-cyhalothrin	0.051 b±0.01	0.04 b±0.01	0.021 c±0.01	0.037 c±0.01
Hexythiazox	0.10 a±0.03	0. 091 a± 0.02	0. 083 a±0.02	0.091 ab±0.02
Fenpyroximate	0.094 a±0.02	0.082 a±0.02	0.061 b±0.01	0.079 ab±0.02
Thiamethoxam	0.061 b±0.01	0.050 b±0.01	0.031 c±0.01	0.068 b±0.02
control	0.12 a±0.01	0.099 a±0.02	0.094 a±0.02	0. 104 a±0.002
LSD (0.05)	0.03	0.024	0.022	0.026
F	11.12	11.44	21.55	9.61
P-value	0.001	0.0009	0.0001	0.002
After treated with LC50
Lambda-cyhalothrin	0.021 c±0.01	0.02 b±0.01	0.011 d±0.001	0.017 a±0.004
Hexythiazox	0.099 ab±0.02	0.089 a±0.02	0.064 b±0.01	0.084 ab±0.02
Fenpyroximate	0.080 b±0.01	0.072 a±0.02	0.047 ±0.01	0.066 b±0.02
Thiamethoxam	0.031 c±0.01	0.023 b±0.002	0.020 c±0.01	0.025 c±0.003
control	0.12 a±0.01	0.099 a±0.01	0.094 a±0.02	0.104 c±0.002
LSD(0.05)	0.023	0.041	0.026	0.026
F	33.63	8.22	16.94	20.99
P-value	0.0000	0.003	0.0002	0.0001

• P = Probability; LSD = Least statistical difference. The same letters means no significant difference at 5% level. Each value is the mean of three samples ±SE.

With regard to Esterase activity after treating bee adults with LC₅₀ of tested insecticides (Table 9) data revealed that EST enzyme was significantly decreased after treating with lambda-cyhalothrin recording 0.021, 0.02 and 0.011 μmol/min/mg protein, while thiamethoxam recorded 0.031, 0.023 and 0.020 μmol/min/mg protein, 12, 24 and 48 h of treatment, whereas, there were no significant differences between hexythiazox and fenpyroximate and control. Lambda-cyhalothrin induced the lowest mean of Esterase enzyme as 0.017 μmol/min/mg protein.

Generally, Esterase enzyme activity was decreased after treating bee workers with lambda-cyhalothrin and thiamethoxam at LC₂₅ and LC₅₀ 12, 24 and 48 h of treatment, also EST enzyme activity was decreased as the concentration of pesticide was increased.

4. Discussion

The obtained results are in agreement with Atkins (1992) who found that the toxicity of pyrethroids were ranged from relatively low to highly toxic to honey bees. Abamectin, chlorfenapyr, deltamethrin, and thiamethoxam were the most toxic to bees among the agrochemicals known, either by topical or oral administration (Rhodes and Scott 2006). Also, Thompson (2003), Desneux et al. (2007) and Costa et al. (2014) found that oral administration of thiamethoxam, was highly toxic to A. mellifera as by spraying, furthermore, Tavares et al. (2017) found that thiamethoxam increased the mortality of honeybee. In addition, Abbassy et al. (2020) reported that the neonicotinoid insecticides, thiamethoxam and imidacloprid were the most toxic tested insecticides to bees by feeding technique, recording LC₅₀ values 0.009 and 0.003mg L-1. In addition, Zhao et al. (2022) reported that the uncritical use of neonicotinoids, pyrethroids, chlorantraniliprole, spinosad, flupyradifurone and sulfoxaflor insecticides has led to harmful effects on honey bee growth and development and also, decrease their foraging activity and pollination services. Recently, Motta et al. (2023) reported that lambda-cyhalothrin induced toxic effect for adult Partamona helleri workers. Also, Souza et al. (2024) found that exposure to thiamethoxam in the field led to a minimal impact on population growth or mortality of bees in the colonies, besides that, thiamethoxam caused severe toxicity to immature honeybees, as for the colonies, the harmful effects can be compensated.

With respect to the effect of tested pesticides on the Glutathione S-transferase (GST), Mixed Function Oxidase MFO, Acetyl cholinesterase (AChE), Esterase and Poly Phenol Oxidase (PPO) the obtained results are in agreement with those of Tu et al. (2009) who reported that AChE has been widely used as a biomarker of general exposure to pollutants, especially organophosphate and carbamate pesticides. In addition, Hinton et al. (1995) reported that the activities of GSTs and carboxylesterase enzymes including in the detoxification of a wide range of toxic compounds. In addition, the biochemical and physiological effects of insecticides can render individual honeybees unable to make their duty easily, thus affecting the colony’s performance (Chakrabarti et al., 2020). Also, Badawy et al. (2014) found that carboxylesterase and GST detoxified the low doses of acetamiprid on honey bee adults. Zhu et al. (2017) found that there is a positive correlation between the toxicity of Lambda-cyhalothrin and the activity of GST enzyme. In addition, Roudsari et al. (2022) found that the exposure of honey bee larvae to imidacloprid (0.16 mg a.i./l) increased the mortality of the treated group compared to untreated controls also, disrupt the physiology of honey bees. Moreover, the activity of AChE and GST enzymes were reduced with sub-lethal concentration. Recently, Ibrahim et al. (2023) found that sulfoxaflor exhibited higher activity on GST in A. mellifera. Recently, Choi et al. (2024) found that Lambda-Cyhalothrin may affect the development of honey bees and induced significant changes in detoxification (GST), antioxidative (SOD and CAT), and AChE at sublethal and residue levels.

5. Conclusion

The obtained data revealed that the tested pesticides were toxic to adult workers of honey bee, Apis mellifera after 24, 48 and 72 hrs of treatment. The tested pesticides affected on the detoxification enzymes in honey bees, whether by increasing or decreasing their activity, and thereby reducing the efficiency of this beneficial pollinator. Our results are valuable not only in evaluating the toxicity of common insecticides on honey bees, but also in highlighting the validity of enzymes activity as appropriate indicators for exposure to agrochemicals.

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Publication Dates

• Publication in this collection
  16 May 2025
• Date of issue
  2025

History

• Received
  23 Sept 2024
• Accepted
  06 Feb 2025