Cellular responses in the Malpighian tubules of postica (Latreille, 1807) exposed to low doses of and boric acid.

Rafael Alexandre C. Ferreira Osmar Malaspina (speaker) Elaine C. M. da Silva Zacarin Introduction subtropical - by - They The atrophied meliponines are regions sting typically . are of the social found planet bees in . characterized tropical and 2 Introduction coffee. sunfloweronion,crops assuch melon, and some 1807) is recognizedpollinatorsas of - The specie The Scaptorigona postica Scaptorigona (Latreille, 3 Introduction natural natural habitat reduction reduction of occupation occupation of green the (agrochemical crops) in (agrochemical areas areas regions urban in Intensive Intensive agriculture 5 control for pest used pesticides to exposed S. postica (non - target ) can be be can insect) target 6 4 Introduction - - Boric Acid Acid Boric Fipronil - - Acts metabolism Acts insect on Act in the receptor of gama aminobutyric acid (GABA) acid gamaaminobutyric of receptor in the Act (C 12 (H H 3 4 BO Cl 2 3 F ) 6 N 4 OS) 5 Introduction Excretory system - Malpighian TubulesMalpighian - - Exposure Side Increasing - effects to pesticides of of in in excretion excretory : : rate cells ; . 6 and others proteins

Introduction P. Vasseur, C. Cossu-Leguille, Environment International 28 (2003) 711–717. Introduction AIM: acid. postica mechanisms of toinduced stress by exposure that indicate toxicity adaptationand/or Detect cellular responses tubulesin the Malpighian okr olwdsso boric and fipronil workers to low doses of S. Material and Methods - Preparation of the contaminatedthe diet: of Preparation Acetone Fipronil - - 0.1 fipronil/Kg of µg food Fipronil Daily intake Dailyintake 0.1 ng/fipronil/bee/day 1 1 µg/mL Cândi Cândi + Fipronil 9 Material and Methods - Preparation of the contaminatedthe diet: of Preparation Boric Boric Acid Acetone - Boric Acid Boric - 0.75% acid boric w/w in food Daily intake Dailyintake 0.75 µg/fipronil/bee/day Boric Boric Acid Cândi Cândi + 10 - Toxicological bioassays

Newly emerged bee (4 days old):

- Control Group; - Solvent Control Group; - Fipronil-treated group; - Boric acid-treated group.

Material and Methods Methods and Material 11 Material and Methods kDa kDa Heatprotein).shock fragmentation(70 protein stressHSP70 and the - - - Fixed Morphological and and Morphological Immunohistochemical Ultrastructural Malpighian analysis; tubules histochemical detection of DNA of detection analysis; 12 - Results LT50 using determinated logwas - Bioassays - rank test, Graph test, rank - Pad software 3.0 Prism 13 Results compacted compacted nuclei (arrowhead). 10 Bars: Brush border (dashed arrow) bee fipronil. (A) the Malpighian tubules stained with histological of morphology - itlgclscin f h einMliha uue f the median Malpighian tubules of Histological sections of control group; control group; A (B) treated continuously with0.75% boric acid; C - vdneo rmvlo cellular content (*) the to lumen (L); removal of evidence of L  m B hematoxylin/eosin S.postica (C) treated with0.1 ng / oprsno . Comparison of (A - C). 14 Results ISCDDK kit acid borickit in ISCDDK arrow),10 lumen (L). Bar: C) Bars: 10 fipronilbeeng0.1 treated / with withthe stained Malpighiantubules tubulesMalpighianofthemedian Histologicalofsections Histochemical test: Histochemical A  m. m. C PAS - treated group. treated  L - m. m. Alcian Blue Alcian D) (B). n L Immunodetection of DNA fragmentation, indicative of cell deathDNAofof cell indicativeImmunodetection fragmentation, Feulgen reactionFeulgen Compacted nuclei are observed (arrowhead) and lumen (L). lumen and(arrowhead) observedarenuclei Compacted in Malpighian tubules of tubulesMalpighian in B D F of bees in the control groupthe controlin bees of S. posticaS. S. postica. S. L . A Brush border(dashedBrush - B : Comparison of Comparison : (A) n and beesand 15 - Results (nu). (nu). Brush border (dashed arrow); vacuolation (arrow) and nuclei with(n) nucleoli fipronil (C). Bar: 10 chronically treated with boric acid 0.75% (B)and treated with0.1 ng / bee bees in control the (A); group comparison between the Malpighian tubules of Histological sections stained withBromophenol Blue A n  m nu C n B nu . . Morphological n 16 Results Arrow indicates strong positive reaction.positive strongArrowindicates treatedwithcontinuously the in phosphatasealkaline with conjugatedsecondaryantibody and (Sigma) antibodymonoclonalthe HSP70 using ofprotein, stress cellular (A - C) Histological sections of Malpighian tubules subjected to immunolocalizationto subjectedtubules Malpighian of sections Histological C C A E 0.75% boric acid boric 0.75% (D) functional functional organic cells. groups in Boric acid F D Triggers secondary response = B INITIAL CYTOTOXICEVENT ; bee treated withtreatedbee ; HSP70 HSP70 (Buffer system) – complexes complexes with 0.1 ng/fipronil0.1 control groupcontrol (E). (C) ; 17 Results (F) Detail of nuclei with chromatin compaction. Magnification: 2.500x Magnification:compaction.chromatin withnuclei (F) of Detail 600x.cytoplasm.Magnification: the(c)(nu).(n); Vacuoles/granules in rings nucleoliconcentric arrow)with(dashed nuclei(arrow); portionapicalthe in higherconcentration in(mi) (mv), mitochondriaMicrovilli A, D) of tubulesMalpighianthe of transmissionElectron Control groupControl Mi mv nu ; B, E) B, n Fipronil - treated bees treated nu n ; mv C, F) S. postica S. Boric acid Boric Mi c . - treated bees treated vp nu Mi . n mv 18 Results (vp) with concretions. Magnification: 10.500xMagnification:(vp)concretions. with vacuoleslabyrinthand(lb) ofbasal the depth invagination showing of Basalcells portion 10.500x Magnification:(vs).secretion of vesiclesdilated with microvilli the(mv). apical Note microvilli to associated(Mi)mitochondria tubulesshowing ofMalpighian the Apical portion G, I) of tubulesMalpighianthe of transmissionElectron Control groupControl Mi ; mv H, K) lb Fipronil - treated bees treated Mi ; I, L) S. postica S. mv Boric acid Boric lb vs . - treated bees treated vp . Mi mv lb vs 19 Results (arrow). Magnification: 21.500x(arrow).Magnification: of ribosomesreleaseapparent with (rer),reticulum roughDetailendoplasmic of insecticides. the withintreated bee dense moreare electron (Mi),which mitochondriaDetail of M, P) of tubulesMalpighianthe of transmissionElectron Control groupControl rer Mi ; N, Q) Fipronil - treated bees treated Mi rer ; O, R) O, S. postica S. Boric acid Boric . - treated bees treated Mi . rer 20 Summary and Conclusion - - - - Most typical undergoing In system Fipronil treated Increased - - both of granules) degree compounds And apical In ; features group cells the exposure expression microvilli) excretion inactivation programmed remained played of . of (large necrosis didn`t organization a - treated role process of metabolically increase induce of were process HSP cell cell groups (presence 70 observed in the death, protection of the in active of HSP amount mineralized some boric ; of since 70 chemical : ; dilated buffer acid cells and no - 21 Considerations areas vicinity assessment doses - on understand histochemical - The In bees . addition data of ; of pesticides from sublethal of cultivated to stingless methods this toxicity on research effects fields bees bee are tests, of could S about and/or . low postica important morphological be doses side used in foraging - effects green of in pesticides tools the in in urban risk low and the to 22 THANKS!

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