Luc P. Belzunces

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. 20,000xa0tonnes 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.

Parasite-insecticide interactions: a case study of Nosema ceranae and fipronil synergy on honeybee

In ecosystems, a variety of biological, chemical and physical stressors may act in combination to induce illness in populations of living organisms. While recent surveys reported that parasite-insecticide interactions can synergistically and negatively affect honeybee survival, the importance of sequence in exposure to stressors has hardly received any attention. In this work, Western honeybees (Apis mellifera) were sequentially or simultaneously infected by the microsporidian parasite Nosema ceranae and chronically exposed to a sublethal dose of the insecticide fipronil, respectively chosen as biological and chemical stressors. Interestingly, every combination tested led to a synergistic effect on honeybee survival, with the most significant impacts when stressors were applied at the emergence of honeybees. Our study presents significant outcomes on beekeeping management but also points out the potential risks incurred by any living organism frequently exposed to both pathogens and insecticides in their habitat.

Gut pathology and responses to the microsporidium Nosema ceranae in the honey bee Apis mellifera.

The microsporidium Nosema ceranae is a newly prevalent parasite of the European honey bee (Apis mellifera). Although this parasite is presently spreading across the world into its novel host, the mechanisms by it which affects the bees and how bees respond are not well understood. We therefore performed an extensive characterization of the parasite effects at the molecular level by using genetic and biochemical tools. The transcriptome modifications at the midgut level were characterized seven days post-infection with tiling microarrays. Then we tested the bee midgut response to infection by measuring activity of antioxidant and detoxification enzymes (superoxide dismutases, glutathione peroxidases, glutathione reductase, and glutathione-S-transferase). At the gene-expression level, the bee midgut responded to N. ceranae infection by an increase in oxidative stress concurrent with the generation of antioxidant enzymes, defense and protective response specifically observed in the gut of mammals and insects. However, at the enzymatic level, the protective response was not confirmed, with only glutathione-S-transferase exhibiting a higher activity in infected bees. The oxidative stress was associated with a higher transcription of sugar transporter in the gut. Finally, a dramatic effect of the microsporidia infection was the inhibition of genes involved in the homeostasis and renewal of intestinal tissues (Wnt signaling pathway), a phenomenon that was confirmed at the histological level. This tissue degeneration and prevention of gut epithelium renewal may explain early bee death. In conclusion, our integrated approach not only gives new insights into the pathological effects of N. ceranae and the bee gut response, but also demonstrate that the honey bee gut is an interesting model system for studying host defense responses.

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.

Conclusions of the Worldwide Integrated Assessment on the risks of neonicotinoids and fipronil to biodiversity and ecosystem functioning

The side effects of the current global use of pesticides on wildlife, particularly at higher levels of biological organization: populations, communities and ecosystems, are poorly understood (Kohler and Triebskorn 2013). Here, we focus on one of the problematic groups of agrochemicals, the systemic insecticides fipronil and those of the neonicotinoid family. The increasing global reliance on the partly prophylactic use of these persistent and potent neurotoxic systemic insecticides has raised concerns about their impacts on biodiversity, ecosystem functioning and ecosystem services provided by a wide range of affected species and environments. The present scale of use, combined with the properties of these compounds, has resulted in widespread contamination of agricultural soils, freshwater resources, wetlands, non-target vegetation and estuarine and coastal marine systems, which means that many organisms inhabiting these habitats are being repeatedly and chronically expose...

Earthworm behaviour as a biomarker – a case study using imidacloprid

To determine whether earthworm behaviour can be used as a biomarker for pollutant effects, an experiment was set up using ®Confidor (imidacloprid) as a model pesticide. The burrowing behaviour of two earthworm species ofdifferent ecological types (the anecic Aporrectodea nocturna and the endogeic Allolobophora icterica) was studied using 2D terraria. The activity of two classical biochemical markers (AchE and GST activities) was also measured. Imidacloprid had no effect on the biochemical markers in either earthworm species whatever the concentration (0.01, 0.1 and 1 ppm). In contrast, earthworm behaviour changed dramatically in the presence of imidacloprid. Following one week of exposure to the pesticide at two different concentrations (0.5 and 1 ppm), burrow length, the rate of burrow reuse and the distance covered decreased for both species. We conclude that earthworm behaviour is a sensitive biomarker that can be studied with cheap and simple apparatus such as 2D terraria.

Standard methods for toxicology research in Apis mellifera

Summary Modern agriculture often involves the use of pesticides to protect crops. These substances are harmful to target organisms (pests and pathogens). Nevertheless, they can also damage non-target animals, such as pollinators and entomophagous arthropods. It is obvious that the undesirable side effects of pesticides on the environment should be reduced to a minimum. Western honey bees (Apis mellifera) are very important organisms from an agricultural perspective and are vulnerable to pesticide-induced impacts. They contribute actively to the pollination of cultivated crops and wild vegetation, making food production possible. Of course, since Apis mellifera occupies the same ecological niche as many other species of pollinators, the loss of honey bees caused by environmental pollutants suggests that other insects may experience a similar outcome. Because pesticides can harm honey bees and other pollinators, it is important to register pesticides that are as selective as possible. In this manuscript, we describe a selection of methods used for studying pesticide toxicity/selectiveness towards Apis mellifera. These methods may be used in risk assessment schemes and in scientific research aimed to explain acute and chronic effects of any target compound on Apis mellifera.

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.

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.