Diniz et al. Toxicity and genetic analysis of bees Scaptotrigona bipunctata after contamination with insecticide accephate Scientific Electronic Archives Issue ID: Sci. Elec. Arch. Vol. 13 (8) August 2020 DOI: http://dx.doi.org/10.36560/13820201157 Article link http://sea.ufr.edu.br/index.php?journal=SEA&page=article&o p=view&path%5B%5D=1157&path%5B%5D=pdf Powered by DOAJ, AGRIS, Latindex, Journal TOCs, CORE, Discoursio Open Science, Science Gate, GFAR, CIARDRING, Academic Journals Database and NTHRYS Technologies, Portal de Periódicos CAPES, CrossRef, ICI Journals Master List. Toxicity and genetic analysis of bees Scaptotrigona bipunctata after contamination with insecticide acephate T. O. Diniz¹*, N. C. Pereira¹, W. C. S. Pizzaia¹, A. A. Sinópolis-Giglioli¹, B. G. Silva2, Y. M. Borges3, T. A. 4 Guedes , M. C. C. Ruvolo-Takasusuki¹ 1 Universidade Estadual de Maringá – Departamento de Biotecnologia, Genética e Biologia Celular 2 Escola Superior de Agricultura "Luiz de Queiroz" / USP – Departamento de Ciências Exatas 3 Instituto Federal do Amazonas 4 Universidade Estadual de Maringá – Departamento de Estatística * Author for correspondence: [email protected] ______________________________________________________________________________________ Abstract. The meliponines can be found in tropical and subtropical regions and is observed in the majority of Latin America. Scaptotrigona bipunctata are stingless bees that build colonies that possess between 2,000 and 50,000 individuals. This study aimed to estimate the toxicity of the insecticide acephate after oral and contact contamination in S. bipunctata. Results revealed differences between the two types of contamination and indicated that S. bipunctata is tolerant to acephate since the mortality rate was low. The findings indicated that there were expression changes of isoenzyme esterases after contamination with the insecticide, which was able to partially inhibit almost all of the enzymes identified in this study. In addition, morphological changes were identified in the midgut of the bees in the first 24 hours of contamination. However, after 48 hours it was observed the regeneration of the peritrophic membrane and other structures, contributing to the survival of the insects. It is essential to propose measures to minimize the impact of agrochemicals on pollinators and this study provides support for investigations on pesticide toxicity in stingless bees. Keywords: organophosphate, esterases, midgut ______________________________________________________________________________________ Introduction negative effects on non-target organisms, such as The meliponines, also known as stingless bees, are bees (Desneux et al., 2007). Organophosphates are bees that have atrophied sting, have variable size one of the major classes of insecticides used in pest and belongs to the Apidae family (Silva et al., 2012). control, which, together with carbamates and Scaptotrigona bipunctata belongs to this class of pyrethroids, corresponds to approximately 40% of bees and are found in Bolivia, Paraguay and Peru the pesticide market. This class of insecticides has a (Witter & Nunes-Silva, 2014). In Brazil, this species broad spectrum of action, causing severe damages is generally found in the Pampas, Atlantic Forest to the nervous system of contaminated individuals and Pantanal biomes (Camargo & Pedro, 2013). (Coutinho et al., 2005). It acts by phosphorylating These species possess interesting biological the enzyme acetylcholinesterase, responsible for characteristics and it can be used in managed inactivating the neurotransmitter acetylcholine, pollination (Venturieri et al., 2011) in tropical and blocking its catalytic activity (Bastos et al., 2015). subtropical regions (Slaa et al., 2006). However, Acephate belongs to the organophosphates class there are few studies showing the susceptibility of S. and is used against pests in several crops, such as bipunctata to insecticides (Santos et al., 2016). cotton, peanuts, potatoes, broccoli, citrus, kale, Although the main function of pesticides is to protect cauliflower, cloves, chrysanthemum, beans, agricultural crops against diseases and pests, the tobacco, melons, peppers, cabbage, tomatoes and application of these substances can cause soy (Mapa, 2017). In Brazil, this insecticide is contamination of soils, water and food, and have 8 Diniz et al. Toxicity and genetic analysis of bees Scaptotrigona bipunctata after contamination with insecticide accephate considered moderately toxic, but its use is prohibited was determined considering the size and behaviour in the United States and Europe. of the individuals. The efficiency of these insecticides can be In order to ensure genetic variability and obtain measured by their action under the target and non- genuine toxicological deductions, each experiment target insects, in particular, bees. The isoenzyme was composed of bees from different colonies. Bees esterases are used as indicators or biomarkers for were collected at the entrance of the colonies and pesticides (Dmitryjuk et al., 2014). These transferred to glass bottles (18 cm x 13 cm) and isoenzymes are part of a diverse group of stored in a BOD incubator, maintained in 28 ºC ± 2 hydrolases capable of catalyzing the cleavage and °C and UR 70 ± 10%. Bees were kept under these formation of ester bonds and are widely distributed conditions for 72 hours and dead individuals were in animals, plants and microorganisms. counted after 24, 48 and 72 hours of exposure. Esterases are divided into four classes: For the contact bioassay, 1 mL of the insecticide acetylesterase, (E.C. 3.1.1.6), arylesterases (E.C. were added into the filter paper and left to dry. After 3.1.1.2), carboxylesterases (E.C. 3.1.1.1), and dried, glass bottles were assembled as described cholinesterases, that includes acetylcholinesterases above. For ingestion bioassay, the insecticide was (E.C.3.1.1.7) and pseudocholinesterases (E.C. placed into the food, which consisted of a liquid 3.1.1.8 (Healy et al., 1991). Many of them have a mixture of crystal sugar and water (1:1). To wide range of possible substrates and various guarantee that the bees would feed efficiently small functions in the metabolism of exogenous and pieces of wood were placed into the food recipient to endogenous compounds acting on behaviour and serve as support. development of insects (Montella et al., 2012). In addition, esterases play an important role in Esterases analyzes insecticide resistance (Shin et al., 2016) and may Head and thorax of worker bees were have the ability to metabolize or degenerate homogenized in propylene tubes of 1.5 mL substances before toxic effects (Claudianos et al, containing 60 µL of β- mercaptoethanol solution and 2015). glycerol 10 %. After that, the samples were Morphophysiological analyzes can also be used to centrifuged at 2,000 rpm for 10 minutes at 4° C. study the effects of exposure to agrochemicals PAGE electrophoresis at 12% was conducted with (Oliveira et al., 2014). The majority of ingested Tris-Glicina buffer 0,1 M pH 8,3 at 200 V, for 4 hours insecticides are metabolized in the midgut of the at 4° C. After the electrophoresis, the gels were bees, thus detoxifying enzymes, especially incubated for 30 minutes in 50 mL of sodium esterases (Moreira et al., 2018), can control the phosphate buffer (0.1 M pH 6.2). Soon after, the interaction of insecticides. buffer was discarded and the staining solution (50 Considering the widespread use of mL of 0.1 M sodium phosphate buffer pH 6.2; 0.03 g organophosphates insecticides in crops that attract of α-naphthyl acetate; 0.03 g of β-naphthyl acetate; bees and the consequences for these non-target 0.06 g of Fast Blue RR Salt) were added. organisms this study aimed to find the lethal The gel was incubated until the bands appeared. concentration (LC50) to oral and contact exposure to Finally, the gels were kept in preservative solution, acephate in S. bipunctata, as well as identify composed of 75% acetic acid and 10% glycerol possible morphological variations and expression dissolved in distilled water for at least 24 hours and changes of esterases. digitized. Methods Light microscopy Biological material After contamination with insecticide in the Adult bees of S. bipunctata were obtained concentrations of 5, 6, 7, 8 e 9 µg a.i./ mL for 24, 48 from six colonies located at the Experimental Farm and 72 hours surviving S. bipunctata bees were of Iguatemi, from Universidade Estadual de Maringá anesthetized under low temperature, and the midgut (UEM) (23º 25 'S, 51º 57' O, UEM, Maringá, PR, was dissected in solution (NaCl 0.1 M, Na2HPO4 Brazil) and taken to the Laboratory of Genetics 0.1 M e KH2PO4 0.1 M). Samples were fixed in Animal from the Department of Biotechnology, aqueous Bouin’s solution (picric acid, formaldehyde, Genetics and Cell Biology of UEM. and acetic acid) for 12 h, dehydrated in an alcohol series of increasing concentrations (70%, 80%, Bioassays 90%, and 100%), diaphanized in xylene (100%), The commercial insecticide Orthene® paraffin embedded, and sectioned into 6 lm slices 750BR, Arysta Lifescience, possess 75% of active using a microtome Leica RM 2250. Then, sections ingredient (a.i.). Preliminary tests were made to were spread on glass slides, rehydrated, and determine the sublethal concentrations (contact: 0.1, stained with Hematoxylin and eosin (H/E). The 0.2, 0.3, 0.4 and 0.5 mg a.i./ mL, ingestion: 5, 6, 7, 8 analyses were performed under Olympus light and 9 µg a.i./ mL). microscope and sections were photographed using For each bioassay 270 S. bipunctata bees were a digital camera. collected. Five concentrations of the insecticide were used, in triplicate, containing 15 bees each, plus the control group. The number of bees in each glass 9 Diniz et al. Toxicity and genetic analysis of bees Scaptotrigona bipunctata after contamination with insecticide accephate Data analysis present study, however S. bipunctata were more The normality of the response variable tolerant to acephate than A. mellifera. (mortality) was verified using the following tests: Falco et al. (2010) observed similar effects, Shapiro-Wilk, Cramér-von Mises and Lilliefors, in where A.