Thiamethoxam exposure deregulates short ORF gene expression in the honey bee and compromises immune response to bacteria.

Maximizing crop yields relies on the use of agrochemicals to control insect pests. One of the most widely used classes of insecticides are neonicotinoids that interfere with signalling of the neurotransmitter acetylcholine, but these can also disrupt crop-pollination services provided by bees. Here, we analysed whether chronic low dose long-term exposure to the neonicotinoid thiamethoxam alters gene expression and alternative splicing in brains of Africanized honey bees, Apis mellifera, as adaptation to altered neuronal signalling. We find differentially regulated genes that show concentration-dependent responses to thiamethoxam, but no changes in alternative splicing. Most differentially expressed genes have no annotated function but encode short Open Reading Frames, a characteristic feature of anti-microbial peptides. As this suggested that immune responses may be compromised by thiamethoxam exposure, we tested the impact of thiamethoxam on bee immunity by injecting bacteria. We show that intrinsically sub-lethal thiamethoxam exposure makes bees more vulnerable to normally non-pathogenic bacteria. Our findings imply a synergistic mechanism for the observed bee population declines that concern agriculturists, conservation ecologists and the public.

Using a toxicoproteomic approach to investigate the effects of thiamethoxam into the brain of Apis mellifera.

Neonicotinoids have been described as toxic to bees. In this context, the A. mellifera foragers were exposed to a sublethal concentration of thiamethoxam (LC50/100: 0,0227 ng de thiamethoxam/µL-1 diet), a neurotoxic insecticide, for 8 days; and it was decided to investigate the insecticide effect on the brain by a shotgun proteomic approach followed by label-free quantitative-based proteomics. A total of 401 proteins were identified in the control group (CG); and a total of 350 proteins in the thiamethoxam exposed group (TMX). Quantitative proteomics data showed up 251 proteins with significant quantitative values in the TMX group. These findings demonstrated the occurrence of shared and unique proteins with altered expression in the TMX group, such as ATP synthase subunit beta, heat shock protein cognate 4, spectrin beta chain-like, mushroom body large-type Kenyon cell-specific protein 1-like, tubulin alpha-1 chain-like, arginine kinase, epidermal growth factor receptor, odorant receptor, glutamine synthetase, glutamate receptor, and cytochrome P450 4c3. Meanwhile, the proteins that were expressed uniquely in the TMX group are involved mainly in the phosphorylation, cellular protein modification, and cell surface receptor signalling processes. Interaction network results showed that identified proteins are present in five different metabolic pathways - oxidative stress, cytoskeleton control, visual process, olfactory memory, and glutamate metabolism. Our scientific outcomes demonstrated that a sublethal concentration of thiamethoxam can impair biological processes and important metabolic pathways, causing damage to the nervous system of bees, and in the long term, can compromise the nutrition and physiology of individuals from the colony.

Acute thiamethoxam toxicity in honeybees is not enhanced by common fungicide and herbicide and lacks stress-induced changes in mRNA splicing.

Securing food supply for a growing population is a major challenge and heavily relies on the use of agrochemicals to maximize crop yield. It is increasingly recognized, that some neonicotinoid insecticides have a negative impact on non-target organisms, including important pollinators such as the European honeybee Apis mellifera. Toxicity of neonicotinoids may be enhanced through simultaneous exposure with additional pesticides, which could help explain, in part, the global decline of honeybee colonies. Here we examined whether exposure effects of the neonicotinoid thiamethoxam on bee viability are enhanced by the commonly used fungicide carbendazim and the herbicide glyphosate. We also analysed alternative splicing changes upon pesticide exposure in the honeybee. In particular, we examined transcripts of three genes: (i) the stress sensor gene X box binding protein-1 (Xbp1), (ii) the Down Syndrome Cell Adhesion Molecule (Dscam) gene and iii) the embryonic lethal/abnormal visual system (elav) gene, which are important for neuronal function. Our results showed that acute thiamethoxam exposure is not enhanced by carbendazim, nor glyphosate. Toxicity of the compounds did not trigger stress-induced, alternative splicing in the analysed mRNAs, thereby leaving dormant a cellular response pathway to these man-made environmental perturbations.

Nanopesticide based on botanical insecticide pyrethrum and its potential effects on honeybees.

Nanotechnology has the potential to overcome the challenges of sustainable agriculture, and nanopesticides can control agricultural pests and increase farm productivity with little environmental impact. However, it is important to evaluate their toxicity on non-target organisms, such as honeybees (Apis mellifera) that forage on crops. The aims of this study were to develop a nanopesticide that was based on solid lipid nanoparticles (SLNs) loaded with pyrethrum extract (PYR) and evaluate its physicochemical properties and short-term toxicity on a non-target organism (honeybee). SLN + PYR was physicochemically stable after 120 days. SLN + PYR had a final diameter of 260.8 ± 3.7 nm and a polydispersion index of 0.15 ± 0.02 nm, in comparison with SLN alone that had a diameter of 406.7 ± 6.7 nm and a polydispersion index of 0.39 ± 0.12 nm. SLN + PYR had an encapsulation efficiency of 99%. The survival analysis of honeybees indicated that PYR10ng presented shorter longevity than those in the control group (P ≤ 0.01). Empty nanoparticles and PYR10ng caused morphological alterations in the bees' midguts, whereas pyrethrum-loaded nanoparticles had no significant effect on digestive cells, so are considered safer, at least in the short term, for honeybees. These results are important in understanding the effects of nanopesticides on beneficial insects and may decrease the environmental impacts of pesticides.

MALDI-imaging analyses of honeybee brains exposed to a neonicotinoid insecticide.

BACKGROUND: Toxicological studies evaluating the possible harmful effects of pesticides on bees are important and allow the emergence of protection and pollinator conservation strategies. This study aimed to evaluate the effects of exposure to a sublethal concentration of imidacloprid (LC50/100 : 0.014651 ng imidacloprid µL-1 diet) on the distribution of certain proteins identified in the brain of Apis mellifera worker bees using a MALDI-imaging approach. This technique enables proteomic analysis of tissues in situ by monitoring the spatiotemporal dynamics of the biochemical processes occurring at a specific time in specific brain neuropils. For this purpose, foraging bees were exposed to an 8-day diet containing a sublethal concentration of imidacloprid corresponding to the LC50/100 . Bees were collected on day 8 of exposure, and their brains analyzed using protein density maps. RESULTS: The results showed that exposure to imidacloprid led to a series of biochemical changes, including alterations in synapse regulation, apoptosis regulation and oxidative stress, which may adversely impair the physiology of these colony bees. CONCLUSION: Worker bee contact with even tiny amounts of imidacloprid had potent effects leading to the overexpression of a series of proteins related to important cellular processes that were possibly damaged by the insecticide. © 2018 Society of Chemical Industry.

Biochemical response of the Africanized honeybee exposed to fipronil.

Bees are recognized worldwide for their social, economic, and environmental value. In recent decades they have been seriously threatened by diseases and high levels of pesticide use. The susceptibility of bees to insecticides makes them an important terrestrial model for assessing environmental quality, and various biomarkers have been developed for such assessments. The present study aimed to evaluate the activity of the enzymes acetylcholinesterase (AChE), carboxylesterase (CaE), and glutathione-S-transferase (GST) in Africanized honeybees exposed to fipronil. The results showed that fipronil at a sublethal dose (0.01 ng/bee) modulates the activity of CaE in all isoforms analyzed (CaE-1, CaE-2, and CaE-3) in both newly emerged and aged bees, and does not affect the activity of AChE or GST. The recovery of the bees after fipronil exposure was also investigated, and these results demonstrated that even the cessation of fipronil ingestion might not lead to complete recovery of individual bees. Even at low doses, fipronil was shown to cause changes in the activity of key enzymes in bees. The possible consequences of these changes are discussed. Environ Toxicol Chem 2017;36:1652-1660. © 2016 SETAC.

Sublethal doses of fipronil intensify synapsin immunostaining in Atta sexdens rubropilosa (Hymenoptera: Formicidae) brains.

BACKGROUND: Although ants are common insects in agricultural ecosystems, few studies have considered how xenobiotics might induce physiological and morphological alterations in these insects. This study aimed to verify the neurotoxic action of sublethal doses of fipronil on the mushroom bodies of brains from the leaf-cutting ant Atta sexdens rubropilosa through immunocytochemistry analysis for the protein synapsin. RESULTS: The LD50 value was established as 1.42 ng ant(-1), and the sublethal doses used were LD50/10 and LD50/100. Synapsin labelling was more evident in the brains extracted from ants exposed to the insecticide, specifically in the regions of glia in the mushroom bodies, compared with the control group. It was possible to measure the intensity of emitted fluorescence in the areas of the mushroom bodies, and a statistical test showed differences between the control group and the treatment group. CONCLUSION: Thus, it is concluded that sublethal doses of the insecticide fipronil intensified synapsin immunostaining, suggesting an increased release of neurotransmitters, which may be linked to neurotoxicity and overexcitation. These sublethal doses may have two different effects: compromising the operation and maintenance of the colony and leading to the establishment of resistance in insects.

Virus present in the reproductive tract of asymptomatic drones of honey bee (Apis mellifera l.), and possible infection of queen during mating.

Virus particles and viral inclusions were detected by transmission electron microscopy examination of sections of the seminal vesicles and mucus gland of asymptomatic young drones from colonies of Apis mellifera lightly infested by Varroa mite. In the mucus gland the infection was found in the muscular sheath and epithelium, while in the seminal vesicle in cells of the outer serosa. Isolated viral particles were also observed in the hemolymph occupying the intercellular spaces of the muscular sheath fibers. In the muscle the virus appeared as polygonal crystalloid inclusions, while in the epithelium mainly inside cytoplasmic vesicles. The infected cells apparently are not damaged. The virus particles are present in the hemolymph and forming more mature structures, as crystalloids, in the muscle. This suggests that the virus is liberated in the body fluid and infects the tissues penetrating the cells through endocytosis. The presence of virus in mucus gland epithelial vesicles raise the possibility of its transference to the gland secretion and therefore, to the semen.

Ultrastructural modifications in the venom glands of workers of Apis mellifera L. (Hymenoptera: Apidae) promoted by topical application of juvenile hormone.

The present study analyzed, the influence of the treatment with juvenile hormone on the ultrastructure of Apis mellifera L. workers' venom glands. Newly emerged workers received topical application of 1 microl of juvenile hormone diluted in hexane, in the concentration of 2 microg/pl. Two controls were used; one control received no treatment (group C1) and other received topical application of 1 microl of hexane (group C2). The aspect of the glandular cells, in not treated newly emerged workers, showed that they are not yet secreting actively. Cellular modifications happened according to the worker age and to the glandular area considered. The most active phase of the gland happened from the emergence to the 14th day. At the 25th day the cells had already lost their secretory characteristic, being the distal area the first to suffer degeneration. The treatment with juvenile hormone and hexane altered the temporal sequence of the glandular cycle, forwarding the secretory cycle and degeneration of the venom gland.

Ultrastructural modifications in the venom glands of workers of Apis mellifera L. (Hymenoptera: Apidae) promoted by topical application of juvenile hormone

The present study analyzed, the influence of the treatment with juvenile hormone on the ultrastructure of Apis mellifera L. workers' venom glands. Newly emerged workers received topical application of 1 æl of juvenile hormone diluted in hexane, in the concentration of 2 æg/æl. Two controls were used; one control received no treatment (group C1) and other received topical application of 1 æl of hexane (group C2). The aspect of the glandular cells, in not treated newly emerged workers, showed that they are not yet secreting actively. Cellular modifications happened according to the worker age and to the glandular area considered. The most active phase of the gland happened from the emergence to the 14th day. At the 25th day the cells had already lost their secretory characteristic, being the distal area the first to suffer degeneration. The treatment with juvenile hormone and hexane altered the temporal sequence of the glandular cycle, forwarding the secretory cycle and degeneration of the venom gland.

O presente estudo analisou, através de estudos ultra-estruturais a influência do tratamento com hormônio juvenil sobre as glândulas de veneno de operárias de Apis mellifera L. Para tanto, operárias recém-emergidas receberam aplicação tópica de 1æl de hormônio juvenil, na concentração de 2 æg/æl, sendo usado o hexano como veículo. Foram feitos dois controles, um que não recebeu nenhum tipo de tratamento (grupo C1) e o outro que recebeu aplicação tópica de 1 æl de hexano (grupo C2). O aspecto das células glandulares, em operárias recém-emergidas, mostra que estas não estão ainda secretando ativamente. Observa-se que alterações celulares ocorrem de acordo com a idade da operária e da região glandular considerada no controle C1. Assim, a fase de secreção mais ativa da glândula ocorre entre a emergência e os 14 dias de idade; aos 25 dias as células já perderam sua característica secretora, sendo a região distal a primeira a sofrer degeneração. Os tratamentos com hormônio juvenil e com hexano alteram a cronologia do ciclo glandular, antecipando o início da secreção e da degeneração da glândula.