Effects of Plant Protection Products on Biochemical Markers in Honey Bees

: Plant protection products (PPPs) are pesticides that protect crops and ornamental plants. PPPs include primarily insecticides, herbicides, and fungicides. Bees’ contact with PPPs can cause immediate death or, in sublethal dose, may affect their physiology and/or behavior. Understanding the effect of PPPs’ sublethal doses is especially important. Contact with a sublethal dose of PPPs generally allows the bee to return to the hive, which may expose the whole colony to the harmful substance. Biochemical changes may affect colony condition, health, and performance. Most of the research on the biochemical effects of PPP in honey bees focuses on insecticides and among them neonicotinoids (especially imidacloprid). The vast majority of research is carried out on Apis mellifera workers. A small part of the research has been conducted on drones and queens. Pesticides, including fungicides and herbicides, may alter antioxidant defense, detoxiﬁcation, gene expression, and immune response of the bee. They affect the drones’ semen quality and metabolic rate of the queen. In this review, the biochemical effect of PPP products in the honey bee was examined, with a focus on the effect on cytochrome P450 monooxygenases, glutathione transferases, and carboxylesterases, which take part in toxin metabolism or the detoxiﬁcation process. PPPs effects on the activity of glutathione peroxidase (GPX), catalase (CAT), superoxide dismutase (SOD), proteases, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and phenoloxidase (PO) are also presented. proteases, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and phenoloxidase (PO) [10,11,18–20]. GPX, CAT, SOD, and GP6D are involved in the antioxidant protective capacity against free radicals. Proteases participate in the digestion of protein chains. AST and ALT take part in amino-acid metabolism and ALP catalyzes the dephosphorylation of various phosphate esters. PO controls melanization of patho-genes. The action of PO is regulated by the activation of zymogen prophenoloxidase (proPO) [21]. Most studies on biochemical effects of the PPPs in honey bees’ concern insecticides, especially neonicotinoids.


Introduction
Plant protection products (PPPs) are pesticides that protect crops and ornamental plants. PPPs include the following primarily: insecticides (for insect control), herbicides (for unwanted-plant control), and fungicides (for fungi control). The use of these compounds increases yield, which is of great importance in food production [1]. The Nobel Prize received by Paul Muller for the invention of the insecticidal properties of dichlorodifenylotrichloroetan (DDT) emphasizes the importance of pesticides for humans [2]. However, non-selective action and bioaccumulation potential are one of the main disadvantages of pesticides [3]. Many of the once widely used pesticides have been withdrawn from the market because of their toxicity to humans and other homeothermic species [4].
More attention is paid to the toxicity of PPPs to honey bees, which as a pollinator has a positive effect on increasing yield and maintaining biodiversity. Bees' contact with PPPs can cause immediate death or, at sublethal doses, may affect their behavior and/or physiology. Understanding the effects of PPPs sublethal doses is especially important. Contact with a sublethal dose of PPPs generally allows the bee to return to the hive, which may expose the whole colony to the harmful substance [5]. Since field studies with colonies of bees are much more complicated and expensive than laboratory experiments, the majority of research is carried out in the laboratory using honey bee workers [6]. 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55].  [30] Beta-cyhalothrin(I) 10,400,000 ppb Orally, acute ↓ Head [20] Oxamyl(I) 68,000 ppb Spraying ⇿ Head and thorax [30] Acephate(I) 168 ppb Orally, 6, 12, 24, 48, 72 h ↓ after 48, 72 h Head and thorax [44] 88,300 ppb Spraying ↓ Head and thorax [30] Sulfoxaflor(I) 58,500 ppb Spraying ↑ Head and thorax [30] Abamectin(I) 33 ppb Orally, acute ↓ Head [20] Propiconazole(F) 24,000 and 60,000 ppb Orally, 1, 5, and 10 days ⇿ in Apis cerana Head [46] Difenoconazole(F) 0.1, 1, and 10 ppb Orally, 10 and 20 days ⇿ Head [26] Tetraconazole(F) 512,500 ppb Spraying ⇿ Head and thorax [30] Glyphosate(H) 0.1, 1, and 10 ppb Orally, 10 and 20 days ↑ after 20 days (0.1 ppb) Head [26] 1,217,500 ppb Spraying ⇿ Head and thorax [30] ↓, decreased activity; ↑, increased activity; ⇿, no effect; I-insecticide; F-fungicide; H-herbicide; data relates to Apis mellifera workers unless otherwise stated. in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55].  [30] Beta-cyhalothrin(I) 10,400,000 ppb Orally, acute ↓ Head [20] Oxamyl(I) 68,000 ppb Spraying ⇿ Head and thorax [30] Acephate (I)   168  in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. Head and thorax [30] ↓, decreased activity; ↑, increased activity;

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in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55].  1,217,500 ppb Spraying ⇿ Head and thorax [30] ↓, decreased activity; ↑, increased activity; ⇿, no effect; I-insecticide; F-fungicide; H-herbicide; data relates to Apis mellifera workers unless otherwise stated.  and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. Head and thorax [30] ↓, decreased activity; ↑, increased activity;

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in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55].  in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. 1,217,500 ppb Spraying ⇿ Head and thorax [30] ↓, decreased activity; ↑, increased activity; ⇿, no effect; I-insecticide; F-fungicide; H-herbicide; data relates to Apis mellifera workers unless otherwise stated. in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. Head [26] 1,217,500 ppb Spraying ⇿ Head and thorax [30] ↓, decreased activity; ↑, increased activity; ⇿, no effect; I-insecticide; F-fungicide; H-herbicide; data relates to Apis mellifera workers unless otherwise stated.
in Apis cerana Midgut [46] ↓, decreased activity; ↑, increased activity; R REVIEW 5 of 14 in drones' semen while decreasing protein content [18,19]. The exposition of young queens to thiacloprid or clothianidin resulted in a reduction in the total hemocyte number and the proportion of active and differentiated hemocytes. Moreover, thiacloprid (200 or 2000 ppb) weakened the antimicrobial activity of the hemolymph and melanization response [55]. Head [26] 1,217,500 ppb Spraying ⇿ Head and thorax [30] ↑, increased activity; ⇿, no effect; I-insecticide; F-fungicide; H-herbicide; data relates to Apis s otherwise stated. , no effect; I-insecticide; F-fungicide; data relates to Apis mellifera workers unless otherwise stated.

Introduction
In addition to neonicotinoids, a frequently used group of insecticides are pyrethroids. Pyrethroids are derivatives of chrysantemic acid, similar to the naturally occurring pyrethrins produced by the flowers of Chrysanthemum L. and Tanacetum L. [56]. Dried flowers of Tanacetum cinerariifolium secrete natural pyrethrins with insecticidal properties [57,58]. Pyrethroids, which are more persistent agents than naturally occurring pyrethrin, cause the prolongation of the opening of sodium channels in the nerve cells of insects, which in turn results in disturbances in the conduction of nerve impulses. It results in excitation, exhaustion, and subsequent paralysis and death of insects and mites [58]. Studies of the biochemical effect in honey bees involved deltamethrin, lambda-cyhalothrin, and beta-cyhalothrin [30,44,48,59].
Another group of PPPs are carbamate insecticides. They blocked the activity of cholinesterases and disturb the functioning of the nervous system. In turn, they result in uncontrolled movement, paralysis, and eventually death [60]. Studies of the biochemical effect in honey bees involved carbaryl and oxamyl [28,30,48].
The organophosphate insecticides form a large group of PPPs. They disturb the action of acetylcholine. A compound that originates from the metabolism of organophosphate binds the acetylcholinesterase. Thus, the deactivation of acetylcholine in the synaptic cleft is not possible, which in turn results in overexcitation followed by paralysis and death. Studies of the biochemical effect in honey bees involved chlorpyriphos and acephate [30,44,61,62].
Sulfoxaflor (sulfoximine insecticide) and abamectin (avermectin insecticide) are pesticides further involved in studies of the biochemical effect in honey bees [20,30]. Similar to the neonicotinoids, sulfoxaflor is a nicotinic acetylcholine receptor agonist. Sulfoxaflor binding causes uncontrolled nerve impulses resulting in muscle tremors followed by paralysis and death [30]. Abamectin is the product of Streptomyces avermitilis fermentation. It binds to the glutamate-gated chloride channels that are found in insect neurons and muscle cells. It results in paralysis and death [63].

Introduction
Fungicides and herbicides are considered much less toxic to bees than insecticides. For this reason, only a few studies on the biochemical effects in honey bees involved these agents. The biochemical effect in honey bees was studied on triazole fungicides (propiconazole, tetraconazole, and difenoconazole), strobilurin fungicide (picoxystrobin), and the herbicide glyphosate.

Fungicides and Herbicides Used in Plant Protection
After 10 days of the exposition, fungicide difenoconazole (orally, 1 ppb) altered the activity of GST and decreased the activity of G6PD in Apis mellifera. The fungicide tetraconazole in spray (512,500 ppb) and orally (84 ppb) did not affect esterase, GST, and AChE activity [30,44]. The herbicide glyphosate (the concentration of 0.1 ppb) increased AChE activity after 20 days (Table 1) and GST after 10 days while the dose of 1 ppb altered GST activity after 10 and 20 days (Table 2) and decreased glucose-6-phosphate dehydrogenase after 10 days [26]. Glyphosate in spray (1,217,500 ppb) did not affect esterase, GST, and AChE activity [30] (Tables 1 and 2). The fungicide picoxystrobin (orally, 18 ppb) did not affect the hemocyte number in Apis mellifera [51]. The fungicide propiconazole (orally, 2400 and 6000 ppb) had no significant effect on GST and AChE activity but changed P450 activity in Apis cerana [46].

Introduction
Bees, especially near crops, have contact with many PPPs at the same time. Various PPPs can react with each other and change their properties. The best-known example of an interaction between pesticides is the synergic effect of ergosterol-biosynthesis-inhibiting (EBI) fungicides and pyrethroid insecticides. Prochloraz, an active ingredient in EBI fungicides, increases the toxic effect of pyrethroids [64]. Studies on PPPs mixtures on honey bees' physiology contribute to enhancing knowledge about the PPPs effects. However, there is still little research on this topic.

Mixtures of Plant Protection Products
In Apis mellifera, the mixture of acephate and oxamyl had less impact on esterase and GST activity than a single pesticide application. On the other hand, the effect of acephate in the composition with clothianidin, tetraconazole, glyphosate, lambda-cyhalothrin, or chlorpyriphos did not differ significantly from treatment with a single pesticide [44]. A mixture of imidacloprid with clothianidin or tetraconazole increased esterase activity. The effect of these mixtures was stronger than a single pesticide treatment. Imidacloprid in combination with lambda-cyhalothrin, oxamyl, sulfoxaflon, or glyphosate did not affect AChE, esterase, and GST in a different manner than a single pesticide [30]. Lambdacyhalothrin and abamectin reduced the toxicity of thiamethoxam. The mixture of these pesticides had less impact on PPO, AChE, and GST activity than a single pesticide treatment. In addition, a combination of thiamethoxam and beta-cyhalothrin had less effect on ALP activity than single pesticide administration [20]. In Apis cerana mixture of propiconazole and acetamiprid caused an increase (after 1 day) or a decrease (after 10 days) in P450 activity [46].

Introduction
PPPs may alter detoxification, antioxidant, and immune-related biochemical mechanisms in honey bees, which impact the functioning of their entire organism. Many studies confirmed that pesticides caused an increase or decrease in enzyme activity and changed the content of some crucial substances (e.g., ATP, proteins, and glutathione) [10,11,20,[24][25][26][27][28][29][30]37,41,42,46,47,52,61,62]. However, the reference values were not estimated [65]. There are many limitations in the interpretation and comparison of the results of biochemical studies on honey bees. Workers differed in age in an individual study (physiological parameters and sensitivity to pesticide change with the bee's age [65,66]). In addition, various materials are collected for analysis (e.g., hemolymph, head, and intestines) while the content and/or activity of indicators may vary in different tissues and organs [28][29][30]. Pesticides used in studies had a wide range of concentrations or doses; they were applied in different manners and for various exposure times, which is also an important issue in interpreting the results.

Effects on Detoxification and Antioxidation
Changes in the activity of detoxification enzymes may result in the accumulation of harmful substances in the honey bee's body. Lower activities of the ALP, ALT, and AST enzymes in bees may impair, among others, the ATP synthesis, Krebs cycle, oxidative phosphorylation, and beta-oxidation. Disorders in the antioxidant system may cause toxic effects through the production of peroxides and free radicals, causing oxidative damage to all cell components. The damage to proteins, lipids, and DNA is particularly severe for the cell.

Effects on Immunity
Changes in hemocyte numbers may contribute to weakened phagocytosis and encapsulation response. Phagocytosis allows the engulfing of pathogens and infected cells, which reduced the spread of infectious agents in organisms. During encapsulation, the bound hemocytes isolate pathogens and neutralize them by anoxia, toxic reactive oxygen species, or starvation [67]. Alteration of the transcription factor NF-κB can cause disturbance in inflammatory responses [68]. Changes in the activity of proteases may result in the poor extracellular or intracellular digestion of proteins, disruption in processes of zymogen activation, the reduced release of hormones and proteins, and hindered translocation across membranes. It may weaken the immunity and resistance to varroosis and nosemosis [69,70]. An especially important function of proteases (more precisely serine proteases) is the activation of the proPO cascade, which is responsible for melanization and sclerotization of the cuticle and participates in immunological processes [71,72].

Effects on Behavior
Changes in AChE activity after pesticide exposure can indicate malfunction of the nervous system. AChE hydrolyzes Ach, which is a major neurotransmitter associated with learning in the insect brain [73]. Changes in AChE activity may affect the learning ability and memory [73][74][75]. Disorders can impact, among others, navigation skills and olfactory learning which are necessary for searching the food and returning to the hive [76,77]. Moreover, alteration in AChE activity may affect grooming behavior, motor function, and cause abdominal spasms [78].

Effects on Individual Development
Serine proteases participate in regulatory cascade pathways. Changes in protease activity may affect individual development [79]. Royal jelly produced in the hypopharyngeal glands of nurse bees contains trace amounts of ACh, which are necessary for the proper larval development [80]. Changes in the level of AChE may result in developmental impairments [80,81].

Effects on Colony Strength
All of the mentioned changes at the molecular level may cause dysregulation in the functioning of the bee's organism, which may manifest itself in behavioral changes, disease resistance, development, production of brood, and finally the weakening of the colony condition and performance. Although most of the studies involved honey bee workers, PPPs are not indifferent to queens and drones [10,18,19,25,29,32,33,47,48,55]. PPPs altered the activity of detoxification, antioxidant, and immune-related enzymes and reduced sperm concentration and viability, which altogether can result in reduced quality and quantity of the bee brood, thereby weakening the bee colony. A strong colony with a sufficient number of individuals is able to provide the right amount of food and take care of the microclimate and hygiene in the hive.

Conclusions
The activity of individual enzymes after treating bees with PPPs differs depending on the dose, method of administration, duration of exposure, and type of the sample for analysis. PPPs may disturb the honey bees' physiology and their effects can vary even if they belong to the same chemical group. Most studies on the biochemical effects of PPPs on honey bees focus on insecticides, especially neonicotinoids, and among them imidacloprid. Cyano-substituted neonicotinoids (acetamiprid and thiacloprid), pyrethroids, and other insecticides are less common subjects of research on the honey bees' biochemical markers. However, results showed that these insecticides affects honey bee biology. The biochemical effects of fungicides and herbicide (glyphosate) in honey bees were the subject of several studies. Although they are considered less toxic, fungicides and herbicides may affect honey bees. The vast majority of research is carried out on Apis mellifera. It is worth emphasizing that results indicated that different physiological mechanisms occurred in Apis mellifera and Apis cerana. Therefore, findings on Apis mellifera physiology should not always be applied to Apis cerana. A small part of the research involves drones and queens.