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Non-Target Inhibition of Antioxidant Enzymes in Honey Bees (A. mellifera and A. florea) Upon Pesticide Exposure

Corresponding Author: Mohammad Ali Al-Deeb Department of Biology, United Arab Emirates University, AlAin, UAE Email: m_aldeeb@uaeu.ae.ae Abstract: Understanding how different insecticides influence bee health is very essential. The aim of this work was to evaluate enzyme kinetics and inhibition by insecticides in Apis mellifera and Apis florea. Tested enzymes showed variable affinity to substrates. In A. florea, glutathione Stransferase (GST) showed significantly higher affinity for its 1-Chloro-2,4Dinitrobenzene (CDNB) substrate than in A. mellifera. Carbosulfan caused the highest inhibition to acetylcholinesterase (AChE) in both tested species in comparison with chlorpyrifos, dimethoate and pirimiphos-methyl. Our results show that exposure to insecticides is harmful to honey bees and results in negative effects on enzyme activity.


Introduction
Apis mellifera and Apis florea are two important plant pollinators and producers of honey in the United Arab Emirates and many other countries. Foraging honey bees are often exposed to insecticides while they are collecting nectar and pollen. In addition, honey bees are affected by Varroa mites, microsporidian parasites like Nosea apis, bacterial diseases such as American foulbrood and viral diseases for example deformed wing virus. Most risk assessment studies examining honey bees have focused mainly on the direct acute exposures to pesticides from spray drifts. However, it should be noticed that honey bees are also exposed to sublethal insecticide concentrations in the form of residues in nectar and pollen. The effects of insecticides on beneficial insects and honey bees can be deleterious and possibly lethal (Stapel et al., 2000). Many studies have shown that the pollen and nectar collected by honey bees and brought back to the hive contain significant concentrations of residues of different pesticides (including insecticides) (Greenpeace, 2014;Choudhary and Sharma, 2008;Chauzat et al., 2006;Pohorecka et al., 2012). Nectar insecticide residues are lower than pollen residues (Dively and Kamel, 2012;Choudhary and Sharma, 2008). Sanchez-Bayo and Goka (2014) have shown that pyrethroid and neonicotinoid insecticides pose the highest risk to bees by contact exposure with contaminated pollen. However, in many cases, the synergism of fungicides with the two above-mentioned classes of insecticides can result in much higher risks regardless of the low concentrations of their combined residues (Sanchez-Bayo and Goka, 2014). Additionally, there is concern when using systemic insecticides because these chemicals can translocate from the soil into the pollen and nectar of plants and as a result, they could be ingested by honey bees and other pollinators (Stoner and Eitzer, 2012). Furthermore, studies have shown that foliar-applied treatments and insecticides applied using drip irrigation during flowering can result in high concentrations of pesticide residues in the pollen and nectar of treated plants (Dively and Kamel, 2012). Therefore, it is not surprising that insecticide residues can be detected in honey. A study reported that insecticide residues were found in honey collected by both domesticated and wild bees. It was indicated that more apiary honeys were found to be contaminated with organochlorines and organophosphates at higher concentrations than honeys produced by wild Apis species (Khan et al., 2004).
It is well-established that upon exposure to various toxins and pollutants, insects have several mechanisms, including induction of detoxifying enzymes, to counteract the toxic effects of pesticides. Detoxifying enzymes, such as esterases, play roles in pesticide synergism and resistance (Ishaaya, 1993). Additionally, increased expression of detoxifying enzymes can provide insects with tolerance to some insecticides, such as pyrethroids (Kostaropoulos et al., 2001). In addition, several factors can induce the detoxifying enzymes in insects (Lee, 1991;Yu, 2004); for example, microsomal epoxidase and GST have been shown to be induced by cruciferous host plants, such as cabbage and their allelochemicals, in diamondback moth larvae (Yu and Hsu, 1993). The induction of detoxifying enzyme activities can provide metabolic resistance against insecticides in many insect species (Li et al., 2007). Studies have shown that GST and oxidase play roles in resistance in selected insecticides. Insecticide resistance primarily occurs due to changes in insect metabolic enzymes or to development of insecticideinsensitive target sites in the insect nervous system (Damayanthi and Karunaratne 2005). Qualitative and/or quantitative changes in GSTs and monooxygenases have been linked to increased insecticide metabolism. In a study, Yu (2004) noted that the decreased toxicity of carbaryl, permethrin and indoxacarb was most likely due to enhanced detoxification of these pesticides by microsomal oxidases and GSTs, which are involved in insecticide metabolism. Unlike the metabolic enzymes, which require high specificity, a high reaction rate and fine tuning by metabolites to carry out their functions, the detoxifying enzymes are slow, have a broad specificity and are essentially unidirectional catalysts that are able to react with a wide spectrum of xenobiotics that cells may encounter (Tang and Chang, 1996). GST enzymes have been reported to play a key role in the detoxification mechanism for insecticides and hence, they may contribute to insecticide resistance in economically important target insect species in diverse agricultural systems (Dou et al., 2006). GST is believed to be crucial because it is a major enzyme involved in phase II detoxification. Furthermore, significant alterations of the total and specific activities of this enzyme were noted during development of the branchiopod Artemia salina (Papadopoulos et al., 2004).
In the UAE, most of the A. mellifera honey bees are imported from Egypt and they differ from the European honey bee strains by having relatively higher tolerance to summer temperatures. Unlike A. mellifera bees that are kept in Langstroth hives, the A. florea honey bees live in open nests and small colonies that are not maintained by beekeepers. The aims of this in vitro study were (1) to measure enzyme kinetics in A. mellifera and A. florea and (2) to study enzyme inhibition by insecticides.

Insecticide Preparation
All insecticide active ingredients were first dissolved in absolute ethanol then diluted in distilled water to attain the appropriate concentrations.

Enzyme Preparation
Adults of A. mellifera and A. florea were homogenized manually (with pestle and mortar) in phosphate buffer for the GST, esterase and oxidase enzyme assays. For the AChE assay, heads of A. mellifera and A. florea adults were homogenized manually in phosphate buffer. All homogenates were centrifuged at 16,500 xg for 10 min at 4°C as described by Smirle (1990). The protein concentrations of insect homogenates were determined according to the method of Bradford (1976) using bovine serum albumin as a standard.

Enzyme Kinetic Parameters GST
The GST activity toward the CDNB substrate was determined according to Habig et al. (1974). Briefly, 250 μL of diluted enzyme preparation, 150 μL of 8.13 mM GSH and 10 μL of CDNB with a range of final concentrations (0.54, 0.27, 0.13, 0.068, 0.034, 0.017 mM) were mixed with 490 μL of 0.1 M potassium phosphate buffer (pH 7.2) and 10 μL CDNB. The nonenzymatic reaction of CDNB and GSH measured without homogenate served as a control. The absorbance was recorded at 340 nm for the CDNB substrate using a spectrophotometer every 2 min for 10 min and converted into nmol CDNB conjugated/min/mg protein with extinction coefficient ε340nm = 9.6 mM 1 cm 1 (Wu et al., 2008;Dou et al., 2006).

General Esterase
Esterase activity was assayed using 1-NA (1-naphthyl acetate) as a substrate as described by Damayanthi and Karunaratne (2005). A volume of 250 μL of diluted homogenates, 300 μL of 7.8 mM Fast Blue B and 10 μL of 1-NA with different concentrations (0.1, 0.026, 0.013, 0.0064 and 0.00322 mM) were mixed with 340 μL of 0.1 M potassium phosphate buffer (pH 7.2). The control was a non-enzymatic reaction, which contained no homogenate. The absorbance was recorded at 620 nm for the α-NA substrate using a spectrophotometer every 2 min for 10 min and converted into nmol α-NA conjugated/min/mg protein with extinction coefficient ε620nm = 2.222 mM 1 cm 1 (He, 2003).

General Oxidase
Oxidase activity was obtained using 3,3,5,5-Tetra-Methylbenzidine (TMBZ) as a substrate as described by Tiwari et al. (2011). The assay was run as follows: 100 μL of the homogenate, 100 μL of TMBZ of different concentrations (0.184, 0.092, 0.045, 0.022, 0.011 and 0.005 mM) and 25 μL of 3% H2O2 were mixed with 675 μL of 0.25 M of diluted sodium acetate buffer (pH 5). The buffer was diluted to avoid the blockage of the substrate position on the enzyme by sodium ions. The control was a non-enzymatic reaction, which contained no homogenate. The absorbance was recorded at 620 nm for the TMBZ substrate using a spectrophotometer every 2 min for 10 min and converted into nmol TMBZ conjugated/min/mg protein with extinction coefficient ε620nm = 3.9×10 4 mM 1 cm 1 (Josephy et al., 1982).

AChE
A volume of 33.3 μL of homogenate was added with 15 μL of an insecticide at the concentrations listed in the esterase inhibition assay. The mixture was incubated for 10 min at room temperature. Then, 199.3 μL of a mixture (10 μL of 0.33 mM DTNB and 189.3 μL of 0.1 M potassium phosphate buffer (pH 7.2)) was added and 4 μL of 1.057 mM ATCh was then added. The whole mixture was incubated for another 45 min at room temperature. The control was a non-enzymatic reaction, which contained no homogenate. The absorbance was recorded at 414 nm for the ATCh substrate using a microplate reader. The change in absorbance was measured as an endpoint reaction (Ellman et al., 1961).

General Esterase
A volume of 83.3 μL of homogenate was added, followed by 15 μL of an insecticide with the following concentrations: (carbosulfan (0.310 nM), chlorpyrifos (0.437 nM), pirimiphos-methyl (0.773 nM) and dimethoate (0.95 μM). The mixture was incubated for 10 min at room temperature, after which 120 μL of a mixture [3.33 μL of 0.1 mM 1-NA +116.6 μL of 0.1 M potassium phosphate buffer (pH 7.2)] was added and 100 μL of 7.8 mM Fast Blue B was then added. The whole mixture was incubated for another 10 min at room temperature. The control was a non-enzymatic reaction, which contained no homogenate. The absorbance was recorded at 620 nm for 1-NA using a microplate reader. The change in absorbance was measured as an endpoint reaction (He, 2003).

Kinetics Parameters of Inhibition (Km, Vmax and Ki)
The inhibition kinetics of general esterases and AChE were calculated with carbosulfan, chlorpyrifos and pirimiphos-methyl. The calculations of the kinetics constants (Km, Vmax and Ki) of esterase and AChE were performed using double-reciprocal plots, obtained by plotting 1/v0 vs 1/[S] to determine the intercepts and the slopes of the linear transformation. Ki is the inhibitor constant, which is the concentration required to produce half maximal inhibition. The same esterase and AChE inhibition assay protocols were followed to conduct the experiments in A. mellifera and A. florea using a microplate reader. The concentration of each insecticide was tested with a range of substrate concentrations of 1-NA (0.1, 0.026, 0.013, 0.0064 and 0.00322 mM) and ATCh (1.057, 0.52, 0.26, 0.13, 0.065, 0.037 and 0.016 mM).

Statistical Analysis
Differences between treatments were tested using analysis of variance t test and Analysis of Variance (ANOVA) using SAS software (SAS Institute Inc., Cary, NC, USA). Means were separated in significant ANOVA using Fisher Least Significant Difference (LSD). The significance value was set at P < 0.05. Each enzyme assay was conducted at least three times.

Enzyme Kinetics Km
The GST of A. florea adults showed significantly higher affinity for the enzyme substrate (18.67±0.67 mM) than adults of A. mellifera (24.97±0.52 mM) (Table  1). However, the general esterase enzymes of A. florea showed a significantly lower substrate affinity (0.63±0.026 mM) compared to A. mellifera (0.39±0.020 mM). The oxidase showed no significant differences between A. mellifera and A. florea in terms of substrate affinity (33.3±1.70 and 26.62±2.57 mM, respectively).

Vmax
Significant differences were found in the Vmax values between adults of A. mellifera and A. florea for GST (195±18.77 and 130±15.53 nmol/min, respectively) and general esterases (693.9±37.53 and 830.5±22.57 pmol/min, respectively) (Table 1). However, the difference between the two species was not significant for the oxidase (49.6±2 and 56.7±1 nmol/min, respectively).

Kinetics Parameters of Inhibition AChE
The presence of 0.310 nM carbosulfan in the reaction mixture resulted in an inhibition of AChE activity in A. mellifera and A. florea. Carbosulfan decreased the Vmax by 51% and increased the Km by 61% in A. mellifera. However, carbosulfan decreased the Vmax by 61% and increased the Km by 79% in A. florea compared with control values in the absence of the insecticide (Table 3). The Ki and Ki' of 0.071±0.005 and 0.290±0.017 nM in A. mellifera and 0.028±0.002 and 0.197±0.010 nM in A. florea, respectively, were calculated for AChE inhibition.
The inclusion of 0.437 nM chlorpyrifos in the reaction for both A. mellifera and A. florea led to a significant inhibition in AChE activity. Compared with control values, chlorpyrifos decreased the Vmax by 44%, while the Km was almost the same in A. mellifera. However, the Vmax decreased by 68% and the Km increased by 74% in A. florea (Table 3 Mean values within one row followed by the same letter are not significantly different from each other according to t test, (P > 0.05). GST = Glutathione S-Transferase, AChE = Acetylcholinesterase. Km = Substrate concentration at which the rate was half of maximum velocity (Vmax); * Units of Vmax are in pmol/min. ** Units of specific activity are in pmol/min/mg protein.   --0.397±0.0250 0.642±0.0520 0.505±0.0550 AChE = Acetylcholinesterase. Km = Substrate concentration at which the rate was half of maximum velocity (Vmax). Ki = Inhibitor constant, which is the concentration required to produce half maximum inhibition Similarly, when 0.773 nM of pirimiphos-methyl was added to the reaction it caused inhibition in general esterase activity for both species. In A. mellifera, the Km decreased by 78% and the Vmax also decreased by 70%. Moreover, the Ki and Ki' values of A. mellifera are 0.112±0.006 and 0.336±0.023 nM, respectively (Table  3). In A. florea, the Km increased by 78% and the Vmax was decreased by 61%. In addition, the Ki and Ki' values for the inhibition of general esterase activity in A. florea are 0.067±0.002 and 0.505±0.055 nM, respectively.

Enzyme Kinetics
The results of the current study show that the A. florea GST has a higher affinity for its substrate (1.3fold) than the GST of the A. mellifera and this is probably a result of species differences or the exclusive feeding of A. florea on wild plants unlike A. mellifera bees, which receive some artificial feeds such as sugar syrups by beekeepers. Additionally, the results demonstrate that the A. mellifera general esterase enzymes has a higher affinity (1.6-fold) for the substrate compared to the general esterase of A. florea. In general, the differences in substrate affinities could be related to interspecific differences. Furthermore, insect species have different dietary needs and show differences in body size and this might be the case in the current study where A. florea is much smaller than A. mellifera. Vanhaelen et al. (2004) performed a study on the purification and characterization of Glutathione Stransferases (GSTs) from two syrphid flies (Syrphus ribesii and Myathropa florea). Their results showed that the specific activity of GST toward CDNB was 1.7-fold higher for S. ribesii than M. florea. With regard to kinetics, M. florea had higher Km values than those from S. ribesii, with values being 2.6, 1.4 and 2-fold higher for GSH, CDNB and DNIB, respectively, indicating differences among species. Also, the Vmax values were higher for S. ribesii, with ratios of 3.2, 5.3 and 1.8 for GSH, CDNB and DNIB, respectively, compared to M. florea.

Enzyme Inhibition
In this study, the tested insecticide concentrations were lower than the ones applied on plants in the field. Carbosulfan and pirimiphos-methyl caused higher inhibition in AChE and general esterase enzymes in both A. mellifera and A. florea. This indicates that these two honey bee species are at risk in the fields treated with carbosulfan and pirimiphos-methyl. These results agree with a published laboratory bioassay showing that carbosulfan was the most toxic insecticide to A. mellifera (Husain et al., 2014). Another study showed that carbosulfan is toxic to A. mellifera (Akca et al., 2009). In fact, pesticide damage becomes more aggravated when bees visit different plants during their foraging trips and therefore, they potentially are exposed to mixtures of pesticides. It was reported that honey bees travel more than 5-6 km and even longer distances to collect pollen and nectar (Hagler et al., 2011;Visscher and Seeley 1982).

Enzyme Inhibition Kinetics
In the current study, insecticides were tested to investigate their potential role in inhibiting AChE and general esterase activities. Generally, carbosulfan, chlorpyrifos and pirimiphos-methyl inhibited AChE and esterase. Detoxifying enzymes and target alteration are equally important mechanisms of insecticide degradation (Pasay et al., 2009). AChE is of interest because it is the target enzyme for organophosphate and carbamate insecticides in the central nervous system (Wang et al., 2004) and its role in cholinergic synapses is essential for life. Several studies have been performed on insect AChE to explore the relationship between AChE alteration and insect resistance (especially to carbamates and organophosphates) (Chai et al., 2007). Price (1988) explained that there are many factors that may affect the susceptibility of AChE to specific insecticides, such as cuticle penetration and metabolic processes. Therefore, AChE sensitivity does not necessarily indicate that insects will be sensitive to a particular chemical when it is used as an insecticide. Substrate inhibition at a high concentration is a normal phenomenon for AChE and is most likely due to the binding of excess substrate to the peripheral (allosteric) anionic site to form an enzymesubstrate complex (Hai-Hua et al., 2005). Carbamate insecticides inhibit AChE by adding a carbamyl group on the enzyme, which completely inactivates the enzyme (Leibson and Lifshitz, 2008). Moreover, organophosphate compounds inhibit cholinesterases (Kamanyire and Karalliedde, 2004), leading to the accumulation of acetylcholine. The inhibition of AChE causes overstimulation of nicotinic and muscarinic acetylcholine receptors (Sogorb and Vilanova, 2002).
The poisoning of A. mellifera arising from the agricultural use of pesticides has been monitored long time ago. In the United Kingdom the measurement of bee head AChE inhibition was used as an indicator of organophosphate insecticide poisoning (Westlake et al., 1985). In 1978, there were widespread reports of serious poisoning associated with honey bees foraging on oilseed rape to which triazophos (organophosphates) had been applied. Investigation of these incidents showed that AChE inhibition was apparently reversible after triazophos poisoning. Additionally, Suh and Shim (1988) reported that GST (CDNB conjugation) activity was significantly induced by diazinon and moderately induced by permethrin (Suh and Shim, 1988). Westlake et al. (1985) reported that lethal insecticide treatment of bees with most carbamate and organophosphate compounds resulted in significant AChE inhibition. In the current study, pirimiphos-methyl was the most potent inhibitor of the esterase enzymes in both A. mellifera and A. florea. Furthermore, the presence of carbosulfan, chlorpyrifos and pirimiphos-methyl in the incubation mixture with the enzymes at different substrate (ATCh and 1-NA) concentrations for AChE and esterase, respectively, exhibited mixed inhibition patterns. However, chlorpyrifos exhibited a non-competitive inhibition because the Km value was not affected. Noncompetitive inhibition is considered a mixed inhibition. Generally, this indicates that the insecticide directly binds to the AChE and esterase active site and allosteric site as well, reducing the concentration of free enzyme available for 1-NA/ATCh binding. This potentially allows the insecticide to persist in a honey bee for a longer period of time because of the lower activity of detoxification achieved by AChE and esterase. He (2003) conducted a study using a continuous spectrophotometric assay for the determination of diamondback moth esterase activity. His results illustrated that the 1-NA exhibited a competitive inhibition toward azadirachtin. However, the current study is different from the work of He (2003) because the inhibiting insecticide was a plant extract (azadirachtin). As a result, they obtained competitive inhibition patterns while mixed and non-competitive inhibitions were observed in the present study. In addition, the current study provided the parameters of the inhibition kinetics for AChE and general esterase in A. mellifera and A. florea in response to exposure to carbosulfan, chlorpyrifos and pirimiphos-methyl.

Conclusion
This study sheds light on four enzymes in A. mellifera and A. florea. It advances our knowledge and understanding about effects of insecticides on honey bees at low concentrations in the environment. No significant differences occurred in the specific activities of GST, general esterase and oxidase between A. mellifera and A. florea. Carbosulfan and pirimiphosmethyl caused higher inhibition to AChE in both A. mellifera and A. florea. Overall, the tested insecticides demonstrated variable inhibition rates and patterns against the four enzymes, which revealed some of the negative effects of insecticides on honey bees. Thus, there is a need for developing new generations of agricultural pesticides that are safer on honey bees. In addition, further studies are warranted to evaluate effects of pesticides on A. florea in particular because it is a wild species and its numbers cannot be augmented by beekeepers unlike A. mellifera, which is kept in hives maintained by beekeepers.