Honey bee (Apis mellifera ligustica) acetylcholinesterase enzyme activity and aversive conditioning following aluminum trichloride exposure.

BACKGROUND: Aluminum is the third most prevalent element in the earth's crust. In most conditions, it is tightly bound to form inaccessible compounds, however in low soil pH, the ionized form of aluminum can be taken up by plant roots and distributed throughout the plant tissue. Following this uptake, nectar and pollen concentrations in low soil pH regions can reach nearly 300 mg/kg. Inhibition of acetylcholinesterase (AChE) has been demonstrated following aluminum exposure in mammal and aquatic invertebrate species. In honey bees, behaviors consistent with AChE inhibition have been previously recorded; however, the physiological mechanism has not been tested, nor has aversive conditioning. RESULTS: This article presents results of ingested aqueous aluminum chloride exposure on AChE as well as acute exposure effects on aversive conditioning in an Apis mellifera ligustica hive. Contrary to previous findings, AChE activity significantly increased as compared to controls following exposure to 300 mg/L Al3+. In aversive conditioning studies, using an automated shuttlebox, there were time and dose-dependent effects on learning and reduced movement following 75 and 300 mg/L exposures. CONCLUSIONS: These findings, in comparison to previous studies, suggest that aluminum toxicity in honey bees may depend on exposure period, subspecies, and study metrics. Further studies are encouraged at the moderate-high exposure concentrations as there may be multiple variables that affect toxicity which should be teased apart further.

Physiological effects of gamma irradiation in the honeybee, Apis mellifera.

Terrestrial ecosystems are exposed to various kinds of pollutants, including radionuclides. The honeybee, Apis mellifera, is commonly used in ecotoxicology as a model species for evaluating the effects of pollutants. In the present study, honeybees were irradiated right after birth for 14 days with gamma rays at dose rates ranging between 4.38 × 10-3 and 588 mGy/d. Biological tissues (head, intestine and abdomen) were sampled at D3, D10 and D14. Ten different physiological markers involved in nervous (acetylcholinesterase (AChE)), antioxidative (catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-transferase (GST)), immune system (phenoloxidase (PO)) and metabolism (carboxylesterases (CaEs) and alkaline phosphatase (ALP)) were measured. Univariate analyses were conducted to determine whether each individual biomarker response was positively or negatively correlated with the dose rate. Then, multivariate analyses were applied to investigate the relationships between all the biomarker responses. Although no mortality occurred during the experiment, several biomarkers varied significantly in relation to the dose rate. Globally, the biomarkers of antioxidant and immune systems decreased as the dose rate increased. Reversible effects on the indicator of the neural system were found. Concerning indicators of metabolism (carboxylesterases), variations occurred but no clear pattern was found. Taken altogether, these results help better understand the effects of ionizing radiation on bees by identifying relevant physiological markers of effects. These results could improve the assessment of the environmental risk due to ionizing radiation in terrestrial ecosystems.

Systemic insecticides (neonicotinoids and fipronil): trends, uses, mode of action and metabolites.

Since their discovery in the late 1980s, neonicotinoid pesticides have become the most widely used class of insecticides worldwide, with large-scale applications ranging from plant protection (crops, vegetables, fruits), veterinary products, and biocides to invertebrate pest control in fish farming. In this review, we address the phenyl-pyrazole fipronil together with neonicotinoids because of similarities in their toxicity, physicochemical profiles, and presence in the environment. Neonicotinoids and fipronil currently account for approximately one third of the world insecticide market; the annual world production of the archetype neonicotinoid, imidacloprid, was estimated to be ca. 20,000 tonnes active substance in 2010. There were several reasons for the initial success of neonicotinoids and fipronil: (1) there was no known pesticide resistance in target pests, mainly because of their recent development, (2) their physicochemical properties included many advantages over previous generations of insecticides (i.e., organophosphates, carbamates, pyrethroids, etc.), and (3) they shared an assumed reduced operator and consumer risk. Due to their systemic nature, they are taken up by the roots or leaves and translocated to all parts of the plant, which, in turn, makes them effectively toxic to herbivorous insects. The toxicity persists for a variable period of time-depending on the plant, its growth stage, and the amount of pesticide applied. A wide variety of applications are available, including the most common prophylactic non-Good Agricultural Practices (GAP) application by seed coating. As a result of their extensive use and physicochemical properties, these substances can be found in all environmental compartments including soil, water, and air. Neonicotinoids and fipronil operate by disrupting neural transmission in the central nervous system of invertebrates. Neonicotinoids mimic the action of neurotransmitters, while fipronil inhibits neuronal receptors. In doing so, they continuously stimulate neurons leading ultimately to death of target invertebrates. Like virtually all insecticides, they can also have lethal and sublethal impacts on non-target organisms, including insect predators and vertebrates. Furthermore, a range of synergistic effects with other stressors have been documented. Here, we review extensively their metabolic pathways, showing how they form both compound-specific and common metabolites which can themselves be toxic. These may result in prolonged toxicity. Considering their wide commercial expansion, mode of action, the systemic properties in plants, persistence and environmental fate, coupled with limited information about the toxicity profiles of these compounds and their metabolites, neonicotinoids and fipronil may entail significant risks to the environment. A global evaluation of the potential collateral effects of their use is therefore timely. The present paper and subsequent chapters in this review of the global literature explore these risks and show a growing body of evidence that persistent, low concentrations of these insecticides pose serious risks of undesirable environmental impacts.

Effects of neonicotinoids and fipronil on non-target invertebrates.

We assessed the state of knowledge regarding the effects of large-scale pollution with neonicotinoid insecticides and fipronil on non-target invertebrate species of terrestrial, freshwater and marine environments. A large section of the assessment is dedicated to the state of knowledge on sublethal effects on honeybees (Apis mellifera) because this important pollinator is the most studied non-target invertebrate species. Lepidoptera (butterflies and moths), Lumbricidae (earthworms), Apoidae sensu lato (bumblebees, solitary bees) and the section "other invertebrates" review available studies on the other terrestrial species. The sections on freshwater and marine species are rather short as little is known so far about the impact of neonicotinoid insecticides and fipronil on the diverse invertebrate fauna of these widely exposed habitats. For terrestrial and aquatic invertebrate species, the known effects of neonicotinoid pesticides and fipronil are described ranging from organismal toxicology and behavioural effects to population-level effects. For earthworms, freshwater and marine species, the relation of findings to regulatory risk assessment is described. Neonicotinoid insecticides exhibit very high toxicity to a wide range of invertebrates, particularly insects, and field-realistic exposure is likely to result in both lethal and a broad range of important sublethal impacts. There is a major knowledge gap regarding impacts on the grand majority of invertebrates, many of which perform essential roles enabling healthy ecosystem functioning. The data on the few non-target species on which field tests have been performed are limited by major flaws in the outdated test protocols. Despite large knowledge gaps and uncertainties, enough knowledge exists to conclude that existing levels of pollution with neonicotinoids and fipronil resulting from presently authorized uses frequently exceed the lowest observed adverse effect concentrations and are thus likely to have large-scale and wide ranging negative biological and ecological impacts on a wide range of non-target invertebrates in terrestrial, aquatic, marine and benthic habitats.

Honeybee biomarkers as promising tools to monitor environmental quality.

The aim of this study was to distinguish the impacts of two different anthropogenic conditions using the honeybee Apis mellifera as a bioindicator associated with a battery of biomarkers previously validated in the laboratory. Both the urban (RAV, Ravine des Cabris) and semi-natural (CIL, Cilaos) sites in La Reunion Island were compared in order to assess the impacts of two types of local pollution using the discriminating potential of biomarkers. Hives were placed at the CIL and RAV sites and honeybees were collected from each hive every three months over one year. Honeybee responses were evaluated with respect to several biochemical biomarkers: glutathione-S-transferase (GST), acetylcholinesterase (AChE), alkaline phosphatase (ALP) and metallothioneins (MT). The results showed a significant difference between the localities in terms of GST, AChE and ALP activities, as regarding midgut MT tissue levels. Compared to the CIL site, ALP and MT tissue levels were higher at the RAV site, although AChE activity was lower. GST displayed more contrasted effects. These results strongly suggest that the honeybees based in the more anthropized area were subjected to sublethal stress involving both oxidative stress and detoxification processes with the occurrence of neurotoxic pollutants, amongst which metals were good candidates. A classification tree enabled defining a decision procedure to distinguish the sampling locations and enabled excellent classification accuracy (89%) for the data set. This field study constitutes a strong support in favour of the in situ assessment of environmental quality using honeybee biomarkers and validates the possibility of performing further ecotoxicological studies using honeybee biomarkers.

Hysteresis of insect acetylcholinesterase.

Pre-steady-state catalytic properties of insect acetylcholinesterase (AChE, EC 3.1.1.7) were studied with the neutral substrate N-methylindoxylacetate. Kinetics of soluble Apis mellifera and Drosophila melanogaster AChE forms showed lags (v(i)=0) before reaching the steady-state. Results were interpreted in terms of slow equilibrium between two conformational states E and E' of insect AChE. Hysteresis of insect AChE has been pointed out for the first time. The hysteretic behaviour was found to depend on the NMIA concentration and the nature of the enzyme. The maximum induction times (tau(max)) to reach the steady-state were 800 and 1000s with soluble AChE from A. mellifera and D.melanogaster, respectively. The orders of magnitude of the tau(max) were high and similar to human AChE and BuChE.

Honeybee Apis mellifera acetylcholinesterase--a biomarker to detect deltamethrin exposure.

The purpose of this study is to investigate the possibility to use acetylcholinesterase (AChE) as a biomarker of exposure to deltamethrin insecticide in the honeybee, Apis mellifera and to test its reliability in the presence of other contaminants, as carbamate insecticide. Joined actions of deltamethrin (pyrethroid) and pirimicarb (carbamate), alone or in association, are investigated on AChE activity in surviving and dead honeybees, with a special focus on the relative proportions of its membrane and soluble forms. At the 0.5X dose (12.5 ng of deltamethrin and/or 2.5 microg of pirimicarb per bee), the residual tissue AChE activity in dead bees was 78% with deltamethrin, 43% with pirimicarb and 33% with dual treatment. In surviving bees, tissue AChE activity represented 250%, and 270% of control AChE activity with deltamethrin and dual treatment, respectively. The analysis of membrane and soluble AChE forms revealed an increase in the soluble form in dead bees after deltamethrin and dual treatment. However, in vitro investigations showed no direct interaction of deltamethrin on soluble and membrane AChE activity. The results suggest that the action of deltamethrin on AChE activity, in honeybee intact organisms, could be due to indirect mechanisms. The duality of AChE response to deltamethrin exposure, exhibited by the possibility of increase (surviving bees) or decrease (dead bees) of its activity has been pointed out for the first time. The important increase in AChE activity in response to deltamethrin, not altered by pirimicarb treatment, suggests that AChE activity could represent a robust biomarker specific to deltamethrin exposure in living bees.

Effects of imidacloprid metabolites on habituation in honeybees suggest the existence of two subtypes of nicotinic receptors differentially expressed during adult development.

Habituation of the proboscis extension reflex (PER) in honeybees (Apis mellifera) is age-dependent. Very young bees (< or =7 days old) require significantly less trials to abolish the response to multiple sucrose stimulations than older bees (> or =8 days old). A nicotinic agonist, imidacloprid, modifies this behaviour by increasing the number of trials in < or =7-day-old bees and by decreasing it in older bees [Neurobiol. Learn. Mem. 76 (2001) 183.]. Here we tested our hypothesis that this effect is associated with a differential expression of two subtypes of nicotinic acetylcholine receptors (nAChRs). By testing the effects of six metabolites of imidacloprid, we show that two of them, olefin and 5-hydroxy-imidacloprid, modify the number of trials needed to habituate the PER in a contrasting manner. Olefin increases the number of trials in both age groups, whereas 5-hydroxy-imidacloprid decreases the number of trials, but only in 8-day-old individuals. We conclude that olefin and 5-hydroxy-imidacloprid are specific agonists of two subtypes of an nAChR that are differentially expressed during adult maturation of young honeybees. Olefin is the agonist of an nAChR expressed in both age groups, whereas 5-hydroxy-imidacloprid is the agonist of a late-onset nAChR that is activated in 8-day-old bees. The implications of this finding for the honeybee biology are discussed.

Discrepancy between acute and chronic toxicity induced by imidacloprid and its metabolites in Apis mellifera.

Imidacloprid is a systemic nitroguanidine insecticide that belongs to the neonicotinoid family. As an agonist of the acetylcholine receptor, it attacks the insect nervous system and is extremely effective against various sucking and mining pests. Oral acute and chronic toxicity of imidacloprid and its main metabolites (5-hydroxyimidacloprid, 4,5-dihydroxyimidacloprid, desnitroimidacloprid, 6-chloronicotinic acid, olefin, and urea derivative) were investigated in Apis mellifera. Acute intoxication by imidacloprid or its metabolites resulted in the rapid appearance of neurotoxicity symptoms, such as hyperresponsiveness, hyperactivity, and trembling and led to hyporesponsiveness and hypoactivity. For acute toxicity tests, bees were treated with doses of toxic compounds ranging from 1 to 1,000 ng/bee (10-10,000 microg/kg). Acute toxicity (LD50) values of imidacloprid were about 60 ng/bee (600 microg/kg) at 48 h and about 40 ng/bee (400 microg/kg) at 72 and 96 h. Out of the six imidacloprid metabolites tested, only two (5-hydroxyimidacloprid and olefin) exhibited a toxicity close to that of imidacloprid. Olefin LD50 values were lower than those of imidacloprid. The 5-hydroxyimidacloprid showed a lower toxicity than imidacloprid with a LD50 four to six times higher than that of imidacloprid. Urea also appeared as a compound of nonnegligible toxicity by eliciting close to 40% mortality at 1,000 ng/bee (10,000 microg/kg). However, no significant toxicity was observed with 4,5-dihydroxyimidacloprid, 6-chloronicotinic acid, and desnitroimidacloprid in the range of doses tested. To test chronic toxicity, worker bees were fed sucrose solutions containing 0.1, 1, and 10 microg/L of imidacloprid and its metabolites for 10 d. Fifty percent mortality was reached at approximately 8 d. Hence, considering that sucrose syrup was consumed at the mean rate of 12 microl/d and per bee, after an 8-d period the cumulated doses were approximately 0.01, 0.1, and 1 ng/bee (0.1, 1, and 10 microg/kg). Thus, all tested compounds were toxic at doses 30 to 3,000 (olefin), 60 to 6,000 (imidacloprid), 200 to 20,000 (5-OH-imidacloprid), and >1,000 to 100,000 (remaining metabolites) times lower than those required to produce the same effect in acute intoxication studies. For all products tested, bee mortality was induced only 72 h after the onset of intoxication.

Contrasting effects of imidacloprid on habituation in 7- and 8-day-old honeybees (Apis mellifera).

We examined the effects of sublethal doses (0.1, 1, and 10 ng per animal) of a new neonicotinoid insecticide, Imidacloprid, on habituation of the proboscis extension reflex (PER) in honeybees (Apis mellifera) reared under laboratory conditions. In untreated honeybees, the habituation of the proboscis extension reflex is age-dependent and there is a significant increase in the number of trials required for habituation in older bees (8-10 days old) as compared to very young bees (4-7 days old). Imidacloprid alters the number of trials needed to habituate the honeybee response to multiple sucrose stimulation. In 7-day-old bees, treatment with Imidacloprid leads to an increase in the number of trials necessary to abolish the response, whereas in 8-day-old bees, it leads to a reduction in the number of trials for habituation (15 min and 1 h after treatment), and to an increase 4 h after treatment. The temporal effects of Imidacloprid in both 7- and 8-day-old bees suggest that 4h after treatment the observed effects are due to a metabolite of Imidacloprid, rather than to Imidacloprid itself. Our results suggest the existence of two distinct subtypes of nicotinic receptors in the honeybee that have different affinities to Imidacloprid and are differentially expressed in 7- and 8-day-old individuals.

Absence of a protective effect of the oxime 2-PAM toward paraoxon-poisoned honey bees: acetylcholinesterase reactivation not at fault.

We investigated the failure of 2-PAM to protect honey bees against poisoning with paraoxon. The protective effect of the oxime 2-PAM against inhibition of acetylcholinesterase (AChE) by paraoxon was estimated in vitro and in vivo and was correlated with the mortality of paraoxon-treated bees. In vitro, 2-PAM protected 90% of AChE activity in the presence of paraoxon and reactivated more than 90% of inhibited AChE. Minor soluble and major membrane-bound forms of bee AChE presented about similar extents of reactivation, but the first order rate constant of reactivation (kobs) of the soluble form is threefold higher than that of the membrane-bound form. However, this difference did not significantly influence the reactivation kinetics of total AChE; the constant kobs of the membrane-bound form reflected that of total AChE. The linear kinetic profile of total AChE reactivation supported the conclusion that there was a single population of reactivatable species. The bimolecular rate constant of reactivation (kr), the dephosphorylation rate constant (k2), and the dissociation constant (Kd) were 646 M-1.min-1, 0.84 min-1 and 1. 30 mM, respectively. In vivo, administration of 2-PAM, after paraoxon exposure, induced a complete protection of AChE activity, but did not elicit any significant effect on mortality in paraoxon-treated bees. The inefficiency of 2-PAM to antagonize paraoxon-induced mortality was not changed by the administration of 2-PAM in pretreatment-therapy and in therapy treatments. These results indicated that the mortality of paraoxon-poisoned honey bees was not due to a lack of AChE reactivation.

Parallel regional quantification of choline acetyltransferase and cholinesterase activity in the central nervous system of an invertebrate (Sepia officinalis).

The present study describes (i) a procedure to dissect the central nervous system of the cuttlefish (Cephalopod) into ten, functionally distinct, anatomical regions of interest and (ii) the parallel measurement of acetylcholine synthesis (choline acetyltransferase) and degradation (cholinesterase) activities. Both aspects (dissection and parallel quantification of acetylcholine synthesis and degradation) could be of great importance for quantitative regional studies in neurochemistry in this animal model, it is interesting to study the cellular and molecular mechanisms involved in learning and aging processes. The parallel quantification of acetylcholine synthesis and degradation applicable to any animal model is pivotal since both enzymes are essential for the cholinergic neurotransmission and may be differentially modulated by specific functions such as learning and aging processes. Furthermore, since choline acetyltransferase and cholinesterase show different localization into the brain, their parallel quantification may underlie the involvement of cholinesterase in non-cholinergic functions, which remain unclear throughout the animal kingdom.

Joint actions of deltamethrin and azole fungicides on honey bee thermoregulation.

The effects of sublethal doses of deltamethrin, a pyrethroid insecticide, and prochloraz and difenoconazole, two azole fungicides, on honey bee thermoregulation were investigated by infrared thermography of honey bees kept at 22 degrees C. Deltamethrin at doses of 0.5 and 1.5 ng/bee did not elicit any significant effect on bee thermogenesis whereas doses of 2.5 and 4.5 ng/bee caused a severe hypothermia. Similarly, prochloraz and difenoconazole did not elicit any significant effect on thermogenesis at doses of up to 850 ng/bee whereas they triggered hypothermia at 1250 ng/bee. When associated with prochloraz or difenoconazole at 850 ng/bee, deltamethrin elicited a joint hypothermia at doses that did not induce a significant effect on thermoregulation when used alone.

Central acetylcholine synthesis and catabolism activities in the cuttlefish during aging.

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities, measured in 10 central regions of young, middle-aged and old cuttlefish, showed a regional heterogeneity but with different age-related distribution patterns. Maximal acetylcholine synthesis and catabolism were observed in the inferior frontal and the optic lobes. Important age-related decreases in ChAT activities were evidenced in most regions, while only moderate variations were found for AChE. Since the superior frontal lobe is involved in visual learning, the dramatic decrease in ChAT activity observed in this lobe (-77%) could be implicated in the learning deficits reported in senescent Sepia.

Modulation of honey bee thermoregulation by adrenergic compounds.

The effects of adrenergic compounds on the thermoregulation of resting honey bees were investigated using a non-traumatizing approach. Bees fed ad lib were treated orally and kept at an ambient temperature of 22 degrees C; the surface thoracic temperature was monitored by infrared thermography. Bees were treated with the adrenergic agonists epinephrine and ephedrine, and with the beta-blocking agent alprenolol. Low doses of adrenergic agonists had no significant effect on thermogenesis, but high doses caused a hypothermia. Alprenolol triggered a dose-dependent hypothermia that was reversed by low doses of epinephrine and ephedrine. Results are discussed with reference to the metabolic and neural adrenergic pathways that may be involved in bee thermoregulation.

Differential response of Apis mellifera acetylcholinesterase towards pirimicarb.

The kinetic analysis of Apis mellifera acetylcholinesterase inhibition by the carbamate pirimicarb showed that native and detergent-solubilized membrane enzyme exhibited slightly different carbamylation kinetics. The acetylcholinesterase form sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC) was carbamylated more rapidly (kapp = 36.4 X 10(-3) min-1) than the PI-PLC-resistant counterpart (kapp = 10.13 X 10(-3) min-1) which had a behavior close to that of the soluble tryptic enzyme (kapp = 11.89 X 10(-3) min-1). A difference in acetylcholinesterase sensitivity towards pirimicarb was also observed between foraging and emerging bees. These results show that the molecular structure, the mode of preparation and the source of acetylcholinesterase from the bee head should be taken into account in accurate toxicological studies.

Acetylcholinesterase from Apis mellifera head. Evidence for amphiphilic and hydrophilic forms characterized by Triton X-114 phase separation.

The polymorphism of bee acetylcholinesterase was studied by sucrose-gradient-sedimentation analysis and non-denaturing electrophoretic analysis of fresh extracts. Lubrol-containing extracts exhibited only one form, which sedimented at 5 S when analysed on high-salt Lubrol-containing gradients and 6 S when analysed on low-salt Lubrol-containing gradients. The 5 S/6 S form aggregated upon removal of the detergent when sedimented on detergent-free gradients and was recovered in the detergent phase after Triton X-114 phase separation. Thus the 5 S/6 S enzyme corresponds to an amphiphilic acetylcholinesterase form. In detergent-free extracts three forms, whose apparent sedimentation coefficients are 14 S, 11 S and 7 S, were observed when sedimentations were performed on detergent-free gradients. Sedimentation analyses on detergent-containing gradients showed only a 5 S peak in high-salt detergent-free extracts and a 6 S peak, with a shoulder at about 7 S, in low-salt detergent-free extracts. Electrophoretic analysis in the presence of detergent demonstrated that the 14 S and 11 S peaks corresponded to aggregates of the 5 S/6 S form, whereas the 7 S peak corresponded to a hydrophilic acetylcholinesterase form which was recovered in the aqueous phase following Triton X-114 phase separation. The 5 S/6 S amphiphilic form could be converted into a 7.1 S hydrophilic form by phosphatidylinositol-specific phospholipase C digestion.