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.
The current study compared the toxicity of different concentrations of boric acid in adult workers of Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae), with toxicological bioassays, and examining the dose-dependent and time-dependent histopathological changes, of the midgut, Malpighian tubules, and postpharyngeal glands. Our results revealed the importance of conducting toxicological bioassays combined with morphological analyses of the organs of ants chronically exposed to insecticides used in commercial ant baits. In vitro bioassays showed that boric acid significantly decreases the survivorship of workers regardless of concentration, whereas the morphological data suggested progressive dose-dependent and time-dependent changes in the organs examined, which were evident in the midgut. The midgut is the first organ to be affected, followed by the postpharyngeal gland and Malpighian tubules. This sequence is in agreement with the absorption pathway of this chemical compound in the midgut, its transference to the hemolymph, possibly reaching the postpharyngeal glands, and excretion by the Malpighian tubules. These progressive changes might be due to the cumulative and delayed effect of boric acid. Our findings provide important information for the understanding of the action of boric acid in ant baits in direct and indirect target organs.
There are several hypotheses about the possible functions of the postpharyngeal gland (PPG) in ants. The proposed functions include roles as cephalic or gastric caeca and diverticulum of the digestive tract, mixing of hydrocarbons, nestmate recognition, feeding larvae, and the accumulation of lipids inside this gland, whose origin is contradictory. The current study aimed to investigate the functions of these glands by examining the protein expression profile of the PPGs of Atta sexdens rubropilosa (Hymenoptera, Formicidae). Mated females received lipid supplementation and their glands were extracted and analyzed using a proteomic approach. The protocol used combined two-dimensional electrophoresis and shotgun strategies, followed by mass spectrometry. We also detected lipid β-oxidation by immunofluorescent marking of acyl-CoA dehydrogenase. Supplying ants with lipids elicited responses in the glandular cells of the PPG; these included increased expression of proteins related to defense mechanisms and signal transduction and reorganization of the cytoskeleton due to cell expansion. In addition, some proteins in PPG were overexpressed, especially those involved in lipid and energy metabolism. Part of the lipids may be reduced, used for the synthesis of fatty alcohol, transported to the hemolymph, or may be used as substrate for the synthesis of acetyl-CoA, which is oxidized to form molecules that drive oxidative phosphorylation and produce energy for cellular metabolic processes. These findings suggest that this organ is specialized for lipid nutrition of adult leaf-cutting ants and characterized like a of diverticulum foregut, with the ability to absorb, store, metabolize, and mobilize lipids to the hemolymph. However, we do not rule out that the PPG may have other functions in other species of ants.
Running title: Long-term low dose Thiamethoxam exposure induces short ORFs AbstractMaximizing crop yields heavily relies on the use of agrochemicals to control insect pests. One of the most widely used insecticides are neonicotinoids. Here, we analysed the impact of sub-lethal chronic long-term exposure to the neonicotinoid Thiamethoxam on gene expression and alternative splicing in brains of Africanized honey bees Apis mellifera. Our results reveal a small number of differentially regulated genes showing a concentration dependent response to two low doses of chronic, 10-day Thiamethoxam exposure. Unexpectedly, most of these genes have no annotated function, but encode short Open Reading Frames (sORFs), a characteristic feature of anti-microbial peptides. Likewise, we find that Thiamethoxam exposure sensitizes bees to infection by non-pathogenic bacteria Bacillus badius and Ochrobactrum anthropi. Moreover, infection with estimated single Serratia marcescens kills bees arguing that Varroa mites may essentially contribute to colony collapse by penetrating the cuticle to spread this pathogen. Our results implicate an altered immune response to Thiamethoxam exposure compromising the immune response leading to a decline in bee populations.Understanding the risk for honey bees requires detailed knowledge of cellular and molecular effects that result from the exposure to an insecticide in order to mitigate negative effects or refine the target specificity towards pest species.Changes in gene expression and processing of RNAs, including alternative splicing, are one of the options available to an organism to respond to environmental perturbations 15,16 . Sub-lethal exposure of xenobiotics can induce modulation of splicing reactions [17][18][19] .Neonicotinoid exposure has been linked to a decline in bee health including a reduction of immune competence 20 ;; 21-23 . Insects do not have antibodies and rely on the innate immune system to fight microbial infections. The best insight on insect immunity stems from studies in the fruit fly Drosophila melanogaster, where cellular and humoral immune responses have been identified 24,25 . The cellular response is mediated by three types of hematopoetic cell lineages 24,26,27 , while the humoral immune system can be split into Toll and Imd pathways [reviewed in 28 ]. The Tollpathway is triggered following an immune challenge by Gram-positive bacteria and fungi, ultimately leading to expression of antimicrobial peptides (AMPs) that are then secreted from the fat body into the hemolymph 24,29,30 . In contrast, the Immune deficiency (Imd) pathway leads to expression of a different set of AMPs after a Gramnegative bacterial infection activates pattern recognition receptors and a complex intracellular signaling cascade 24,25,29 . AMPs are short peptides of 10-100 amino acids and are characterised by an evolutionary dynamism among insect species 31 .We have previously shown that worker-bee larvae in colonies contaminated with the neonicotinoid imidacloprid, have altered expression o...
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.
Although solitary bees represent at least 70% of bee species, most ecotoxicological studies on bees focus on social species and there are only a few species of solitary bees. One of the challenges in developing toxicological studies on solitary bees is the lack of protocols for maintaining these species under laboratory conditions. This study aimed to develop a method to maintain adult individuals of the solitary bee Centris analis under controlled conditions. Six models of cages with adaptations for food identification and resting areas were tested based on cages used for Apis mellifera and Osmia bicornis. With this in mind, we placed one individual of Centris analis in each cage and set the temperature at 28°C. Cage models with survival higher than 50% of individuals were used to measure the influence of photoperiod, training and concentration of food in bee adaptation and longevity. Cages containing a wood cube and an artificial fabric flower supported the survival of 75% of captive bees for longer than 30 days at 28°C. Bee survival rate was higher in the absence of photoperiod. This work showed the importance of an acclimatization period of 3 days for C. analis and described a feasible method for future studies evaluating the effects of pesticide effects on solitary bees under laboratory conditions.
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