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The article focuses on the systemic insecticide fipronil and those which belong to the neonicotinoid family. The increasing global dependance on the prophylactic use of these potent neurotoxic systemic insecticides has raised concerns about their effects on biodiversity and ecosystem services provided by a wide range of affected species and environments.

The One Health approach acknowledges that human health is firmly linked to animal and environmental health. It involves using animals such as bees and other pollinators as sentinels for environmental contamination or biological indicators. Beekeepers noticed intoxications of apiaries located in the vicinity of sheep and cattle farms, which led to the suspicion of bees' intoxication by the products used for livestock: veterinary medicinal products (VMPs) and Biocides, confirmed by laboratory analysis. We review the legal context of VMPs and Biocidal products considering Europe as a case study, and identify shortcomings at the environmental level. We describe the possible ways these products could intoxicate bees in the vicinity of livestock farms. We also illustrate the way they may impact non-target species. The cases of ivermectin and abamectin as VMPs, deltamethrin and permethrin as Biocides are considered as case studies. We show bees can be exposed to new and unrecognized routes of exposure to these chemicals, and demonstrate that their application in livestock farming can affect the survival of pollinators, such as bees. We conclude that: (1) figures on the marketing/use of these chemicals should be harmonized, centralized and publicly available, (2) research should be devoted to clarifying how pollinators are exposed to VMPs and Biocides, (3) toxicity studies on bees should be carried out, and (4) pollinators should be considered as non-targeted species concerning the environmental risk assessment before their marketing authorization. We propose the term "Multi-use substances" for active ingredients with versatile use., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (© 2021 The Authors.)

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.

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.

Systemic insecticides are applied to plants using a wide variety of methods, ranging from foliar sprays to seed treatments and soil drenches. Neonicotinoids and fipronil are among the most widely used pesticides in the world. Their popularity is largely due to their high toxicity to invertebrates, the ease and flexibility with which they can be applied, their long persistence, and their systemic nature, which ensures that they spread to all parts of the target crop. However, these properties also increase the probability of environmental contamination and exposure of nontarget organisms. Environmental contamination occurs via a number of routes including dust generated during drilling of dressed seeds, contamination and accumulation in arable soils and soil water, runoff into waterways, and uptake of pesticides by nontarget plants via their roots or dust deposition on leaves. Persistence in soils, waterways, and nontarget plants is variable but can be prolonged; for example, the half-lives of neonicotinoids in soils can exceed 1,000 days, so they can accumulate when used repeatedly. Similarly, they can persist in woody plants for periods exceeding 1 year. Breakdown results in toxic metabolites, though concentrations of these in the environment are rarely measured. Overall, there is strong evidence that soils, waterways, and plants in agricultural environments and neighboring areas are contaminated with variable levels of neonicotinoids or fipronil mixtures and their metabolites (soil, parts per billion (ppb)-parts per million (ppm) range; water, parts per trillion (ppt)-ppb range; and plants, ppb-ppm range). This provides multiple routes for chronic (and acute in some cases) exposure of nontarget animals. For example, pollinators are exposed through direct contact with dust during drilling; consumption of pollen, nectar, or guttation drops from seed-treated crops, water, and consumption of contaminated pollen and nectar from wild flowers and trees growing near-treated crops. Studies of food stores in honeybee colonies from across the globe demonstrate that colonies are routinely and chronically exposed to neonicotinoids, fipronil, and their metabolites (generally in the 1-100 ppb range), mixed with other pesticides some of which are known to act synergistically with neonicotinoids. Other nontarget organisms, particularly those inhabiting soils, aquatic habitats, or herbivorous insects feeding on noncrop plants in farmland, will also inevitably receive exposure, although data are generally lacking for these groups. We summarize the current state of knowledge regarding the environmental fate of these compounds by outlining what is known about the chemical properties of these compounds, and placing these properties in the context of modern agricultural practices.

Imidacloprid, a new systemic insecticide used as seed-dressing, has been widely used in France since 1994. Its application mode and its efficiency allow a significant reduction in comparison with the usual quantity of chemicals used during pulverising treatment. But the insecticide imidacloprid is suspected to have harmful effects on the pollinators as many bees have died since its introduction. Recent studies have shown that imidacloprid has chronic and sub-lethal toxicities at levels of micro g/kg or less. It was therefore necessary to detect imidacloprid at these levels in soils, plants, flowers, and pollens. With this aim, we characterised the bio-availability of imidacloprid in the environment using a new quantitative analytical method, as a basis for the evaluation of the risk for bees.

The enzyme spectrum of an ectoparasitic mite of the honeybee. Varroa destructor (Anderson and Trueman) was studied using a semi-quantitative method, especially designed for complex samples which have not been purified. Exopeptidases and phosphatases are shown present. A chitinase and enzymes able to transform beta carbohydrates are also present with a large range in the intensity of the reaction. The role of the chitinase can be related to the supply of nutritional needs or/and the piercing and sucking behaviour of the adult parasite. Chitinase activity could be one factor influencing the balance between the parasite and its host.

Androctonin is a highly cationic antimicrobial peptide from scorpion exhibiting a broad spectrum of activities against bacteria and fungi. It contains 25 amino acids including four cysteine residues forming two disulfide bridges. We report here on the determination of its solution structure by conventional two-dimensional (2D) 1H-NMR spectroscopy and molecular modelling using distance geometry and molecular dynamics methods. The structure of androctonin involves a well-defined highly twisted anti-parallel beta-sheet with strands connected by a more variable positively charged turn. A comparison with the structure of tachyplesin I (horseshoe crab) reveals that the amphiphilic character of the protein surface of this homologous peptide is not observed in androctonin. We have undertaken a 200-ps molecular dynamics simulation study on a system including one androctonin molecule and a monolayer of DMPG (1,2-dimyristoylphosphatidylglycerol) lipids. On the basis of this simulation, the first steps of the membrane permeabilization process are discussed.

A series of 9 lactonic lipopeptide biosurfactants was isolated from Bacillus licheniformis IM 1307 as representatives of the lichenysin group and we propose to name them lichenysins G. They were recovered from the culture medium as complex mixtures of molecules having different peptide sequences and different structures of beta-hydroxy fatty acids. Their separation was achieved by a reversed-phase HPLC method leading to eight well-separated compounds. The complete structure of individual isoforms was proposed following the results of amino acid and fatty acid analysis, LSI-MS and 2D NMR spectroscopies. Compared to surfactin, lichenysins G are at least 10 fold more efficient biosurfactants.

Thanatin is the first inducible insect peptide that has been found to have, at physiological concentrations, a broad range of activity against bacteria and fungi. Thanatin contains 21 amino acids including two cysteine residues that form a disulfide bridge. Two-dimensional (2D) 1H-NMR spectroscopy and molecular modelling have been used to determine its three-dimensional (3D) structure in water. Thanatin adopts a well-defined anti-parallel beta-sheet structure from residue 8 to the C-terminus, including the disulfide bridge. In spite of the presence of two proline residues, there is a large degree of structural variability in the N-terminal segment. The structure of thanatin is quite different from the known structures of other insect defence peptides, such as antibacterial defensin and antifungal drosomycin. It has more similarities with the structures of various peptides from different origins, such as brevinins, protegrins and tachyplesins, which have a two-stranded beta-sheet stabilized by one or two disulfide bridges. Combined with activity test experiments on several truncated isoforms of thanatin, carried out by Fehlbaum et al. [Fehlbaum, P., Bulet, P., Chernysh, S., Briand, J. P., Roussel, J. P., Letellier, L., Hétru, C. & Hoffmann, J. (1996) Proc. Natl Acad. Sci. USA 93, 1221-1225], our structural study evidences the importance of the beta-sheet structure and also suggests that anti-Gram-negative activity involves a site formed by the Arg20 side-chain embedded in a hydrophobic cluster.

The solution structure of the anti-mammal and anti-insect LqqIII toxin from the scorpion Leiurus quinquestriatus quinquestriatus was refined and compared with other long-chain scorpion toxins. This structure, determined by 1H-NMR and molecular modeling, involves an alpha-helix (18-29) linked to a three-stranded beta-sheet (2-6, 33-39, and 43-51) by two disulfide bridges. The average RMSD between the 15 best structures and the mean structure is 0.71 A for C alpha atoms. Comparison between LqqIII, the potent anti-mammal AaHII, and the weakly active variant-3 toxins revealed that the LqqIII three-dimensional structure is closer to that of AaHII than to the variant-3 structure. Moreover, striking analogies were observed between the electrostatic and hydrophobic potentials of LqqIII and AaHII. Several residues are well conserved in long-chain scorpion toxin sequences and seem to be important in protein structure stability and function. Some of them are involved in the CS alpha beta (Cysteine Stabilized alpha-helix beta-sheet) motif. A comparison between the sequences of the RII rat brain and the Drosophila extracellular loops forming scorpion toxin binding-sites of Na+ channels displays differences in the subsites interacting with anti-mammal or anti-insect toxins. This suggests that hydrophobic as well as electrostatic interactions are essential for the binding and specificity of long-chain scorpion toxins.

The biosynthesis of bacterial isoleucyl-rich surfactins was controlled by supplementation of L-isoleucine to the culture medium. Two new variants, the [Ile4,7]- and [Ile2,4,7]surfactins, were thus produced by Bacillus subtilis and their separation was achieved by reverse-phase HPLC. Amino acids of the heptapeptide moiety were analysed by chemical methods, and the lipid moiety was identified by beta-hydroxy anteiso pentadecanoic acid by combined GC/MS. Sequences were established on the basis of two-dimensional NMR data. Because conformational parameters issuing from NMR spectra suggested that the cyclic backbone fold was globally conserved in the new variants, structure-activity relationships were discussed in details on the basis of the three-dimensional model of surfactin in solution. Indeed, both variants have increased surface properties compared with that of surfactin, and this improvement is assigned to an increase of the hydrophobicity of the apolar domain favouring micellization. Furthermore, the additional Leu-to-Ile substitution at position 2 in the [Ile2,4,7]surfactin leads to a substantial increase of its affinity for calcium, when compared with that of [Ile4,7]surfactin or surfactin. This effect is assigned, from the model, to an increase in the accessibility of the acidic side chains constituting the calcium binding site. Thus, the propensities of such active lipopeptides for both hydrophobic and electrostatic interactions were improved, further substantiating that they can be rationally designed.

We describe the secondary structure and the overall fold of toxin III from the venom of the scorpion Leiurus quinquestriatus quinquestriatus determined using two-dimensional-1H-NMR spectroscopy. This protein, which contains 64 amino acids and 4 disulfide bridges, belongs to the long-chain toxin category and is highly toxic to both mammals and insects. The overall fold was determined on the basis of 1208 inter-proton-distance restraints derived from NOE measurements and 90 psi, phi dihedral-angle restraints derived from NOE connectivities and 3JNH-alphaH coupling constants using the HABAS program. This fold, which mainly consists of an alpha-helix packed against a small antiparallel three-stranded beta-sheet, and of several turns and loops, is similar to that of other long-chain scorpion toxins. Aromatic and non-polar residues form several patches on the surface of the protein which alternate with patches of charged and polar residues. Such a topology should be important in the interactions of toxin III with sodium channels in membranes. Two weakly constrained loops introduce some flexibility to the structure which could be related to the activity of this toxin. The central core of toxin III is compared with the cysteine-stabilized alpha beta motif (an alpha-helix connected to a beta-sheet through two disulfide bridges) found in insect defensins and plant thionins. Defensins and thionins are small proteins (approximately 40--50 amino acid residues) containing three or four disulfide bridges, respectively. This comparison confirms that the cysteine-stabilized alpha beta motif is a common core to a number of small proteins from different origins and having different activities.

Background: Insect defensin A is a basic 4 kDa protein secreted by Phormia terranovae larvae in response to bacterial challenges or injuries. Previous biological tests suggest that the bacterial cytoplasmic membrane is the target of defensin A. The structural study of this protein is the first step towards establishing a structure-activity relationship and forms the basis for understanding its antibiotic activity at the molecular level., Results: We describe a refined model of the three-dimensional structure of defensin A derived from an extensive analysis of 786 inter-proton nuclear Overhauser effects. The backbone fold involves an N-terminal loop and an alpha-helical fragment followed by an antiparallel beta-structure. The helix and the beta-structure are connected by two of the three disulphide bridges present in defensin A, forming a so-called 'cysteine-stabilized alpha beta' (CS alpha beta) motif. The N-terminal loop, which is locally well defined, can occupy different positions with respect to the other moieties of the molecule., Conclusions: The CS alpha beta motif, which forms the core of the defensin A structure, appears to be a common organization for several families of small proteins with toxic properties. The distribution of amino acid side chains in the protein structure creates several hydrophobic or hydrophilic patches. This leads us to propose that the initial step in the action of positively charged defensin A molecules with cytoplasmic membranes may involve interactions with acidic phospholipids.

Light scattering and 31P-NMR have been used to monitor the effect of the bee-toxin, melittin, on phosphatidylcholine (PC) bilayers of variable acyl chain length (from C16:0 to C20:0). Melittin interacts with all lipids provided the interaction is initiated in the lipid fluid phase. For low-to-moderate amounts of toxin (lipid-peptide molar ratios, Ri > or = 15), the system takes the form of large spheroidal vesicles, in the fluid phase, whose radius increases from 750 A with dipalmitoyl-PC (DPPC) to 1500 A with diarachinoyl-PC (DAPC). These vesicles fragment into small discoids of 100-150 A radius when the system is cooled down below Tc (the gel-to-fluid phase transition temperature). Little chain length dependence is observed for the small objects. Small structures are also detected independently of the physical state of lipids (gel or fluid) when Ri < or = 5 and provided the interaction has been made above Tc. Small discs clearly characterized for DPPC and distearoyl-PC (DSPC) lipids are much less stable with DAPC. However in the long term, all these small structures fuse into large lipid lamellae. Discs are thermodynamically unstable and kinetics of disappearance of the small lipid-toxin complexes increases as the chain length increases in the sense: DAPC >> DSPC > DPPC. Kinetics of fusion of the small discs into extended bilayers is described by a pseudo-first-order law involving a lag time after which fusion starts. Increasing the chain length decreases the lag time and increases the rate of fusion. Formation of both the large vesicles in the fluid phase and the small discs in the gel phase as well as their stability is discussed in terms of relative shapes and dynamics of both lipids and toxin.

When Bacillus subtilis S 499 was grown on a culture medium containing L-alanine as nitrogen source, a mixture of surfactins was obtained. Suitable chromatographic conditions allowed the separation of isoforms. Among these compounds, a new variant of surfactin was isolated and its structure was established by chemical and spectrometric methods, especially by NMR spectrometry. It contains a peptide sequence which differs from that of standard surfactin by the replacement of the L-valine residue by L-alanine residue in position 4. The folding mode of [Ala4]surfactin as deduced from NMR results was compared with that of standard surfactin and the structure/properties relationship issuing from the study of this new isoform is discussed.

The solution three-dimensional structure of the protonated [Leu7]-surfactin, an heptapeptide extracted from Bacillus subtilis, has been determined from two-dimensional 1H-nmr performed in 2H6-dimethylsulfoxide and combined with molecular modeling. Experimental data included 9 coupling constants, 61 nuclear Overhauser effect derived distances, NH temperature coefficients, and 13C relaxation times. Two distance geometry (DISMAN) protocols converged toward models of the structure and the best of them were refined by restrained and unrestrained molecular dynamics (GROMOS). Two structures in accord with the set of experimental constraints are presented. Both are characterized by a "horse saddle" topology for ring atoms on which are attached the two polar Glu and Asp side chains showing an orientation clearly opposite to that of the C11-13 aliphatic chain. Amphipathic and surface properties of surfactin are certainly related to the existence of such minor polar and a major hydrophobic domains. The particular "claw" configuration of acidic residues observed in surfactin gives important clues for the understanding of its cation binding and transporting ability.

A 500 MHz 2D 1H NMR study of recombinant insect defensin A is reported. This defense protein of 40 residues contains 3 disulfide bridges, is positively charged and exhibits antibacterial properties. 2D NMR maps of recombinant defensin A were fully assigned and secondary structure elements were localized. The set of NOE connectivities, 3JNH-alpha H coupling constants as well as 1H/2H exchange rates and delta delta/delta T temperature coefficients of NH protons strongly support the existence of an alpha-helix (residues 14-24) and of an antiparallel beta-sheet (residues 27-40). Models of the backbone folding were generated by using the DISMAN program and energy refined by using the AMBER program. This was done on the basis of: (i) 133 selected NOEs, (ii) 21 dihedral restraints from 3JNH-alpha H coupling constants, (iii) 12 hydrogen bonds mostly deduced from 1H/2H exchange rates or temperature coefficients, in addition to 9 initial disulfide bridge covalent constraints. The two secondary structure elements and the two bends connecting them involve approximately 70% of the total number of residues, which impose some stability in the C-terminal part of the molecule. The remaining N-terminal fragment forms a less well defined loop. This spatial organization, in which a beta-sheet is linked to an alpha-helix by two disulfide bridges and to a large loop by a third disulfide bridge, is rather similar to that found in scorpion charybdotoxin and seems to be partly present in several invertebrate toxins.

Reinvestigation of surfactin, a previously studied peptidolipid surfactant from Bacillus subtilis, by fast-atom-bombardment mass spectrometry and 1H-NMR spectroscopy, as well as by chemical methods, revealed the presence of a closely related second constituent. This new compound, [Val7]surfactin, differs from the known surfactin by the C-terminal amino acid residue which is valine instead of leucine.

The actions of bee venom melittin and delta-lysin from Staphylococcus aureus on membranes have been monitored by solid-state deuterium and phosphorus NMR and shown to differ depending on temperature and on the lipid-to-peptide molar ratio Ri. In the gel phase of phosphatidylcholine model membranes, for lipid-to-peptide ratios Ri greater than 15, melittin induces isotropic lines interpreted as reflecting the presence of small discoidal structures, whereas delta-lysin does not. These small objects are metastable, that is, within a time-scale of hours they return to large lipid bilayers. The kinetics of this process depend on the lecithin chain length. In the fluid phases, at temperatures greater than that of the gel-to-fluid transition Tc, analysis of the quadruplar splittings in terms of chain ordering indicates that both melittin and delta-lysin similarly disorder the membrane. At temperatures above but close to Tc, melittin preferentially orders the center of the bilayer, while delta-lysin promotes ordering throughout the entire bilayer thickness. These effects are interpreted as reflecting different locations of the peptides with respect to the membrane surface. The addition of greater amounts of toxins, Ri = 4, on phosphatidylcholine model membranes induces very small structures irrespective of the temperature in the case of melittin, but only above Tc for delta-lysin. NMR spectral features similar to those characterizing the small fast-tumbling objects with phosphatidylcholine are also observed with egg phosphatidylethanolamine and erythrocyte membranes. The formation of small structures is thus inferred as a general process which reflects membrane supramolecular reorganization.(ABSTRACT TRUNCATED AT 250 WORDS)