Enzymatic responses in the head and midgut of Africanized Apis mellifera contaminated with a sublethal concentration of thiamethoxam.

The increasing use of insecticides, promoted by the intensification of agriculture, has raised concerns about their influence on the decline of bee colonies, which play a fundamental role in pollination. Thus, it is fundamental to elucidate the effects of insecticides on bees. This study investigated the damage caused by a sublethal concentration of thiamethoxam - TMX (0.0227 ng/µL of feed) in the head and midgut of Africanized Apis mellifera, by analyzing the enzymatic biomarkers, oxidative stress, and occurrence of lipid peroxidation. The data showed that the insecticide increased acetylcholinesterase activity (AChE) and glutathione-S-transferase (GST), whereas carboxylesterase (CaE3) activity decreased in the heads. Our results indicate that the antioxidant enzymes were less active in the head because only glutathione peroxidase (GPX) showed alterations. In the midgut, there were no alkaline phosphatase (ALP) or superoxide dismutase (SOD) responses and a decrease in the activity of CaE was observed. Otherwise, there was an increase in GPX, and the TBARS (thiobarbituric acid reactive substances) assay also showed differences in the midgut. The TBARS (thiobarbituric acid reactive substances) assay also showed differences in the midgut. The results showed enzymes such as CaE3, GST, AChE, ALP, SOD, and GPX, as well as the TBARS assay, are useful biomarkers on bees. They may be used in combination as a promising tool for characterizing bee exposure to insecticides.

Sublethal doses of imidacloprid and pyraclostrobin impair fat body of solitary bee Tetrapedia diversipes (Klug, 1810).

Solitary bees present greater species diversity than social bees. However, they are less studied than managed bees, mainly regarding the harmful effects of pesticides present in agroecosystems commonly visited by them. This study aimed to evaluate the effect of residual doses of imidacloprid and pyraclostrobin, alone and in combination, on the fat body (a multifunctional organ) of the neotropical solitary bee Tetrapedia diversipes by means of morphological and histochemical evaluation of oenocytes and trophocytes. Males and females of newly-emerged adults were submitted to bioassays of acute topical exposure. Experimental groups were essayed: control (CTR), solvent control (ACT), imidacloprid (IMI, 0.0028 ng/µL), pyraclostrobin (PYR, 2.7 ng/µL) and imidacloprid + pyraclostrobin (I + P). The data demonstrated that the residual doses applied in T. diversipes adults are sublethal at 96 h. Both oenocytes and trophocytes cells responded to topical exposure to the pesticides, showing morphological changes. In the IMI group, the bee oenocytes showed the greatest proportion of vacuolization and altered nuclei. The pyraclostrobin exposure increased the intensity of PAS-positive labeling (glycogen) in trophocytes. This increase was also observed in the I + P group. Changes in energy reserve (glycogen) of trophocytes indicate a possible mobilization impairment of this neutral polysaccharide to the hemolymph, which can compromise the fitness of exposed individuals. Also, changes in oenocytes can compromise the detoxification function performed by the fat body. This is the first study to show sublethal effects in neotropical solitary bees and highlight the importance of studies with native bees.

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.

Toxicological and histopathological effects of boric acid on Atta sexdens rubropilosa (Hymenoptera: Formicidae) workers.

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.

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.

The Postpharyngeal Gland: Specialized Organ for Lipid Nutrition in Leaf-Cutting Ants.

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 ß-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.

Toxicological and histopathological effects of hydramethylnon on Atta sexdens rubropilosa (Hymenoptera: Formicidae) workers.

The leaf-cut ants are important agricultural pest, because they can cause intense defoliation in plants and destroy large areas cultivated. Although there are several works for the control of these insects by examining the toxicity of natural chemical compounds on various species of ants, few are focused on analyses of morphological changes caused in the affected organs. The aim of this study was to evaluate the effects of hydramethylnon on Atta sexdens rubropilosa workers through toxicological bioassays and morphological analysis of the post-pharyngeal glands, midgut, and Malpighian tubules of these ants. Hydramethylnon dissolved either in acetone (HA) or in a mixture of acetone and soy oil (HAO) was added to the artificial diet at a concentration of 200µg/mL. The workers fed daily with the diet containing hydramethylnon showed higher mortality than the controls, especially when HAO was used. Moreover, light and electron microscopy revealed morphological alterations in the midgut and Malpighian tubules of workers treated with HA, whereas alterations of the post-pharyngeal glands were observed in the HAO-treated group. These results indicated that the presence of soy oil provided an alternate route for the ingestion of the formicide's active ingredient and corroborated previous studies that suggested a role for the post-pharyngeal glands in lipid metabolism. Our findings suggest that the oil may carry hydramethylnon to the gland lumen, resulting in lower quantity of the active ingredient in the intestinal lumen and Malpighian tubules that explains the lower degree of morphological alterations in these structures in the workers treated with HAO. These results may provide insight into the toxicological effects of hydramethylnon on leaf-cutting ants and the use of vegetable oil as an adjuvant in baits to control ants.

On the morphology of the worker immatures of the leafcutter ant Atta sexdens Linnaeus (Hymenoptera: Formicidae).

Leafcutter ants of the genus Atta Fabricius are serious agricultural pests. Morphological studies of immature stages within this group are few, and the data provided for species of considerable importance are usually incomplete. In this study, the immatures of Atta sexdens Linnaeus are described and compared using light and scanning electron microscopy. Only specimens from founding stage colonies (i.e., lacking adult workers) were used. The existence of four larval instars was estimated by a frequency plot of maximum head widths, and the larvae of different instars differed from each other mainly by their bodily dimensions. Worker larvae belonged to two distinct morphological castes: (1) gardeners and nurses and (2) within-nest generalists. The worker larvae described in this study differed from a previous description of the same species by the following traits: the existence of a genal lobe, the number of clypeal hairs, the presence of two hairs on the ninth abdominal somite, the presence of hairs on the anterior surface of the labrum, and the shape of the maxillary palpus. This study provides a comparative analysis of immature stages of A. sexdens that may be relevant to future morphological and biological studies of the Attini.