Cell stress and activity of biomarkers enzymes in Africanized bees Apis mellifera LINEU, 1758 (Hymenoptera, Apidae) exposed to thiamethoxam

Author(s)

Pâmela Decio

• Document type: Doctoral Thesis
• Press: Rio Claro. 2019-06-19.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Rio Claro
• Defense date: 2019-04-15
• Advisor: Osmar Malaspina; Thaisa Cristina Roat

Abstract

Africanized Apis mellifera bees are considered important pollinators in Brazil, since many food crops depend on the pollination by these insects. However, with the growth of agricultural productivity there has been an increase in the use of insecticides for pest control, which also can reach non-target insects. With the action of insecticides as one of the possible causes of the massive death of these pollinators, the studies of the effects of pesticides on bees were highlighted. Given the context, the present study aimed to investigate the effects of a sub lethal dose of thiamethoxam - TMX (0.0227 ng of active ingredient / μl of food), a insecticide of the class of neonicotinoids, in the brain and intestine of Africanized Apis mellifera, through the evaluation of the activity of enzymatic biomarkers of exposure and of oxidative stress and by the occurrence of lipid peroxidation. The level of cell stress was also investigated by the immunostaining of the HSP70 and HSP90 proteins together with the detection of cell death by the TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. The data showed that, in the brain, TMX increased acetylcholinesterase activity (AChE) at 1, 3 and 5 days of exposure, while carboxylesterase (CaE) decreased on the first day and glutathione s-transferase (GST) increased in the fifth day. On the other hand, antioxidant enzymes were less active in the brain, and only glutathione peroxidase (GPX) showed increased activity on the first day of exposure. In the intestine, there was no alkaline phosphatase (FAL) or GST response. But there was a decline in CaE activity on the first and fifth day of contamination. Differently from the brain, antioxidant enzymes were most affected in the intestine with increased superoxide dismutase (SOD) on the fifth and GPX in the third day. The TBARS test only showed an increase in the determination of lipid peroxidation in the intestine after 5 days of exposure. In addition to that, it was possible to observe positive immunoblotting of HSP70 and HSP90 in the brain and intestine in almost every analyzed time (except for HSP90 in the brain in the first 24 hours of exposure), but no cell death was detected in any of the evaluated groups. The generated data indicates that, in general, TMX induces alteration on biomarkers of exposure mainly in nerve tissue while antioxidant enzymes were modulated mostly in the intestine. Furthermore, the results show that the high expression of HSPs 70 and 90 has a protective effect, preventing cell death in the brain and in the intestinal epithelial tissue of bees exposed to the sub lethal dose of neonicotinoid. A second part of the study was conducted at the University of Birmingham (UK) and showed that sub lethal doses of pesticides such as thiametoxam, fungicide carbendazim (CAB) and herbicide glyphosate (GLF) were not sufficient to cause alteration in the alternative splicing pattern of the Dscam and elav genes involved with the formation of the neurons network of the nervous system, as well as no changes were observed for the Xbp1 gene, which, from a different type of splicing, triggers a response that avoids the accumulation of Unfolded Protein Response (UPR) under conditions of stress. These results suggest that the sublethal doses used and the exposure time (1 day) were not sufficient to indicate responses in these 3 genes as biomarkers in A. mellifera. By means of enzymatic assays, HSPs immunostaining and cell death, and molecular biology techniques for the detection of alternative splicing, this study contributes with several data characterizing the effects of neonicotinoid TMX on A. mellifera bees, thus providing information that can support arguments to lead the discussion on the laws regulating the use of these pesticides and the management of these pollinators in agricultural areas. (AU)

• FAPESP's process: 14/23197-7 - Cellular stress and activity of biomarkers enzymes in Africanized honeybees Apis mellifera Lineu, 1758 (Hymenoptera, Apidae) exposed to thiamethoxam
• Grantee: Pâmela Decio Horst
• Support Opportunities: Scholarships in Brazil - Doctorate