27 Imidacloprid is widely used for exterminating harmful insects; however, information 28 regarding its distribution in insects is limited. Herein, we developed a visualization method for 29 imidacloprid in Drosophila melanogaster, by using matrix-assisted laser desorption/ionization 30 imaging mass spectrometry (MALDI-IMS). IMS requires sample cryosections; however, certain 31 challenges prevail in retaining fly morphology in sections owing to their small size and 32 heterogeneous components.

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... herefore, the section preparation method was optimized first, 33 followed by imidacloprid distribution visualized using MALDI-IMS. Using 10% gelatin as an 34 embedding material and 70% ethanol for pretreatment, the gaps between embedding material 35 and D. melanogaster body surface were reduced. The tight adhesion between embedding media 36 and D. melanogaster retains fly morphology in sections. Furthermore, imidacloprid standard 37 was analyzed separately via MALDI and electrospray ionization (ESI) and imidacloprid was 38 converted to guanidine-imidacloprid via laser irradiation. Consequently, imidacloprid 39 distribution in D. melanogaster was successfully visualized using guanidine-imidacloprid as the 40 target peak. 41 Keywords: Matrix-assisted laser desorption/ionization-imaging mass spectrometry, Insecticide, 42 Imidacloprid, Drosophila melanogaster 43 44 45 Analytical Sciences Advance Publication by J-STAGE Introduction 46 47 Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) has 48 been extensively used as the method of choice to obtain spatial information regarding target 49 compounds without labeling. Thus far, IMS technology has been primarily applied for assessing 50 animal tissue in the medical field, including drug distribution or metabolism. 1,2 Recently, IMS 51 visualization targets have expanded towards food materials, 3 plants, 4 and insects, especially 52 Drosophila melanogaster. 5,6 53 D. melanogaster is one of the most important model organisms for neurological and 54 biological studies, and is widely used to study insecticide metabolism and resistance 55 mechanisms. 7,8 Imidacloprid, a neonicotinoid insecticide, is widely used for exterminating 56 harmful insects. However, recently it has also been shown to exert a negative impact on honey 57 bees. 9,10 It serves as a nicotinic acetylcholine receptor agonist in insects; 11 however limited 58 information is available regarding insecticide distribution in insects. Hence, in this study, a 59 visualization method for imidacloprid in D. melanogaster was developed using MALDI-IMS. 60 Though numerous imaging studies using D. melanogaster have been reported, 5,12 certain 61 challenges prevail. One of the major challenges for D. melanogaster imaging is retaining fly 62 morphology in sections owing to their small size and heterogeneous components such as hard 63 external structure with cuticle and soft cells lacking cytoskeleton. In this study, we also suggest 64 an optimized method for obtaining sections wherein fly morphology is retained. 65 We successfully obtained suitable tissue sections via pretreatment with 70% ethanol and 66 10% gelatin as embedding material. With this optimized method, imidacloprid was visualized in 67 D. melanogaster at the target m/z of guanidine imidacloprid (m/z 211.07). Imidacloprid m/z 68 256.06 could not be used as the target m/z for MALDI-IMS as it changed to guanidine 69 imidacloprid via laser irradiation. 70 Analytical Sciences Advance Publication by J-STAGE