Toxicity of A-cypermethrin, Emamectin Benzoate and Imidacloprid on AChE and Antioxidant Enzymes in The Honey Bee (Apis mellifera)

Introduction

Honey bees are considered to be good pollinators of many vegetable and fruit crops. Without adequate populations of bees, the production of these and other crops would be impossible. In addition, bee colonies are maintained for their honey and wax production. Many insecticides used for pest control are toxic to honey bees and therefore they can be used as indicator organisms for environmental pollution with pesticides.

The use of environmental biomarkers in ecotoxicology is becoming a useful routine, and various endpoints have been proposed as valuable tools to assess the effects of environmental chemical contamination. Among the most biomarkers, acetylcholinesterase (AChE) inhibition which are being frequently used both in environmental monitoring and laboratory assays. AChE is an important enzyme for the maintenance of normal nerve function. Inhibition of this enzyme resulting from the irreversible binding at the AChE active site (ester-forming site) leads to the accumulation of acetylcholine in the synapse, resulting in the disruption of normal function (Ibrahim et al., 1998). Other biomarkers are also of great interest such as that related to detoxification potential of the studied organisms. In recent years, several reports have suggested that, insecticides cause oxidative stress, characterized by exposure to excessive reactive oxygen species (ROS). The body has developed several defense mechanisms against oxidative damage. These mechanisms are composed of enzymatic and non-enzymatic systems. The enzymatic mechanism is made of free radical scavengers like catalase (CAT) and the glutathione-depend enzymes such as glutathione-s-transferase (GST) and glutathione peroxidase (GP_X)(Durak et al., 2010).

Malondialdehyde (MDA) is one of the indicators of lipid peroxidation, and it also react with DNA, protein, enzyme and other biomolecules, leading to oxidative damage. Any variability in MDAdeterminations may arise from variability in non-enzymatic chemical events yielding lipid peroxide products. Some of these products may increase or decrease the activities of specific antioxidant enzymes (Janero, 1990).

Therefore, the aim of the present research is to test the in vivo toxicity of α-cypermethrin, emamectin benzoate andimidacloprid against the honey bee (A. mellifera) workers.

MATERIALS AND METHODS

 Honey bee (Apis mellifera)

First cross (Carnilolan* Egyptian) honey bee colonies reared in the apiary of El-Sabhia Research Station, Agriculture Research Center, Ministry of Agriculture, Alexandria, Egypt were used in the present investigation. Bee workers were stored in a temperature controlled chamber in the dark at 25±1.5°C and 65±5% RH where they remained quiet and protected from stress-induced biochemical changes. Bees of age of 3-4 weeks were fed on 50% (W/V) sucrose solution ad libitum according to Sharaf El-Din)1982(.

Chemicals

Acetylthiocholine iodide (ATCI), 5.5'-dithio-bis-2-nitrobenzoic acid (DTNB), reduced glutathione (GSH), 1-chloro-2,4-dinitrobenzene (CDNB); Triton x-100, Folin Ciocalteau phenol reagent and bovine serum albumin (BSA) were purchased from sigma – Aldrich chemical company (Fancy Road, Poole, Dorest, BH12 4QH, England). Other chemicals used either for hatching procedure, toxicity tests or biochemical assays were obtained from BDH Laboratory supplies (Ltd. Limited, Poole, UK).

Tested Insecticides

The insecticides tested were commercial formulations of α-cypermethrin (α-Zed 10% EC) as a, emamectin benzoate (Elector 2% EC) and imidacloprid (Best 25% EC) and these formulations were supplied by Arab Company for the Manufacture of Pesticides and Veterinary Medicines, Egypt.

Toxicity of the tested insecticides

Adult honeybee workers were exposed to a range of concentrations of each of the test compounds (α-cypermethrin, emamectin benzoate and imidacloprid) dispersed directly in 50% sucrose solution. Three replicate test groups, each of ten bees were used in each experiment. Three control batches, each of ten bees, were run in addition to the tested series (control insects were fed on 50% sucrose solution only). The bees were held in an experimental room at a temperature of 25 ± 2ºC and a relative humidity of 50-70%.The mortality was recorded after 24 and 96hrs and compared with control values. If the untreated check (control) group recorded mortality, correction for control mortality is made using Abbott’s formula (Abbott, 1925). Analysis of the mortality data was achieved by appropriate statistical methods (probit analysis) (Finney, 1971). Median lethal concentrations (LC₅₀) and associated 95% confidence limits were calculated.

Half and quarter of LC₅₀ were also calculated for each insecticide from the propit (mortality) / log dose regression equation (y= a + bx), where y= 4.3255 and 3.8497, respectively.

AChE assay

Laboratory strains of nursery honey bee workers were fed on α-cypermethrin, emamectin benzoate and imidacloprid dispersed in 50% sucrose solutions at ¼ LC₅₀ (12.5% mortality), ½ LC₅₀ ( 25% mortality) and LC₅₀ ( 50% mortality) plus an  untreated group as a control. AChE activity was assessed (in vivo) in head of adult honey bee workers. The heads were cut from surviving bees at time end points of     24 hr, placed in eppendorf  tubes, snap frozen in liquid nitrogen and stored at 20ºC for AChE assay. AChE activity was assayed according to Ellman et al.)1961(.

Antioxidant enzymes assay

After 24 hrs of feeding on the tested insecticides (α-cypermethrin, emamectin benzoate and imidacloprid) at the concentration of ¼ LC₅₀, ½ LC₅₀ and LC₅₀ plus untreated group (as a control), GST, CAT, and GP_x were assessed (in vivo) in midgut homogenate of the adult honey bee workers.

Catalase (CAT) was determined according to Aebi (1984), Glutathione-s-transferase (GST) was determined according to Habig et al. (1974), Glutathione peroxidase (GP_X) was measured according to Paglia and Valentine (1967) and Malondialdehyde level (MDA) was estimated according to Sotoh (1978) and Ohkawa et al. ( 1979). All data were expressed as mean ± SD

RESULS and DISCUSIONS

Toxicity of α-cypermethrin, emamectin benzoate and imidacloprid on the honey bee workers (A. mellifera)

Results inTable (1) presented the calculated LC₅₀ and the associated parameters (confidence limits and slope) of each of the tested insecticides against the workers of the honey bee. It is evident from LC₅₀ values of the tested insecticides that emamectin benzoate was the most toxic tested compound tohoney bee workers with LC₅₀ values of 0.275 and 0.184mg/l after 24 and 96 hr time exposure, respectively, followed by α-cypermethrin with LC₅₀ values of 5.411 and 3.267mg/l after 24 and 96 hr of treatment, respectively. Imidacloprid was proved to be the least toxic insecticide with LC₅₀ values of 24.97and 10.98mg/l after 24 and 96 hrs of treatment, respectively.

It was noticed that, the toxicity of the tested insecticides against A. mellifera increased asthe exposure time increased.

Yan-Xia et al. (2008) reported that  methylamino abamectin benzoate 1% EC was extremely toxic to bees with LC₅₀ value of 0.490 mg/l after 48-hrs. The presented results are also in accordance with Laurino et al. (2013). Who found that LD₅₀ value of imidacloprid  at 24 hours was 118 ng/bee, at 48 hours was 90.09 ng/bee and at 72 hours was 69.68ng/bee and they also reported that the toxicity of imidacloprid increased with the increase of the exposure time. Moreover, Hassona and Kordy (2015)proved that certain pesticides (Vertimec^® as a bio-insecticide) was toxic against honey bee forgers. Rasuli et al. (2015)showed that fenpropathrin as a synthetic pyrethroid insecticide had high acute oral toxicity (LC₅₀-24h and LC₅₀-48hrs were 0.54 and 0.30 ppm, respectively).

Table (1). Toxicity of α-cypermethrin, emamectin benzoate and imidacloprid against the honey bees (A. mellifera) workers after 24 and 96hrs of treatment

             )a: lethal concentration causing 50% mortality after 24hr and b: lethal concentration causing 50% mortality after 96hr)

In vivo AChE activity in A. mellifera exposed to α-cypermethrin, emamectin benzoate and imidacloprid for 24hr

The results in Figure (1) show the in vivo effects of the tested insecticides on AChE activityof A. mellifera. The results revealed that imidacloprid at the concentrations of 6.2, 12.5, and 24.97 mg/l (¼ LC₅₀, ½ LC₅₀ and LC₅₀) caused 6.2 16.5, and 23.4% inhibition, respectively. Also, α-cypermethrin caused inhibition percentages of 2.1, 5.1, and 7.5% when it was tested at the concentrations of 1.6, 2.7, and 5.4mg/l, subsequently. On the other hand, emamectin benzoate at the concentrations of 0.07, 0.15, and 0.275mg/l caused 60, 73.2, 82.8% increase of A. mellifera AChE activity, respectively.

These present results are in agreement with those of Bendahau et al. (1999)who reported that cypermethrin slightly inhibit acetylcholinesterase activity on honey bees and this because AChE is not a specific target or site to synthetic pyrethroids. Also, Badiou et al. (2008) used acetylcholinesterase (AChE) as a biomarker (or indicator) of exposure to deltamethrin insecticide in the honey bee, A. mellifera.Moreover, Menessy (2011)revealed that avermectin insecticide (milbemectin) cause in vivo activation of AChE of honey bee head.  In addition, Badiou et al. (2012)observed that there were no response for AChE on honey bees treated with the neonicotinoid insecticide thiamethoxam. Moreover, these results are in accordance with those obtained by Boily et al. (2013) who reported that AChE activity on foraging bees was inhibited in response to a neonicotinoid (imidocloprid).

Figure (1). In vivo effects of α-cypermethrin, emamectin benzoate and imidacloprid on AChE  activity of A. mellifera.

Effects of α-cypermethrin, emamectin benzoate and imidacloprid on antioxidant enzymes activities and MDA level in A. mellifera

Theresults in Table (2) show the in vivo effects of the tested insecticides on certainantioxidant enzymes activities of A. mellifera. Data revealed that there was a significant increase in the catalase (CAT), glutatione-s-transferase (GST) and glutathione peroxidase (GP_x) activities depend on the concentration of insecticides used as compared to the control.

In addition, there was a decrease in the level of Malondialdehyde (MDA) in the case of α-cypermethrin and imidacloprid. On the other hand, emamectin benzoate caused slight elevation (11.1%) in the level of MDA in A. mellifera as compared with control (Table 3).

These results demonstrate that enhanced activities of CAT, GST, and GPx can lead to elimination of ROS in the midgut of honey bees. Also, the increase in CAT, GST, and GPx activities may be an attempt to counteract the increase in MDA level as a defense mechanism by cells against free radicals generation.

The oxidative destruction of lipids acts in a chain reaction to form lipid hydroperoxides, which can decompose to MDA as the end product. The decrease in lipid peroxidation during the treatment with α-cypermethrin and imidacloprid may be due to an increase in antioxidant defense.

These results are in agreement with those of Yu et al. (1984)who found that the insecticidepermethrin significantly stimulated the glutathione-s-transferase in adult worker bees (Apis mellifera L.). Also, Johnson et al. (2006)reported that three pyrethroids (cyfluthrin, lambda-cyhalothrin and tau-fluvalinate) may activate GSTs enzyme in honey bee worker. Moreover, these finding are supported with those of Badiou et al. (2012). They evaluated the development of GST and CAT as enzyme biomarkers of exposure to neonicotinoid xenobiotics such as thiamethoxam in the honey bee, A. mellifera and they found an increase on GST and CAT activities due to such exposure.

Table (2). In vivo effects of α-cypermethrin, emamectin benzoate and imidacloprid on the activity of these three enzymes in treated A.mellifera workers

                     *mean ± SD