Abstract
Apis mellifera is an important bee pollinating native and crop plants but its recent population decline has been linked to the use of pesticides, including fungicides that have been commonly classified as safe for bees. However, many pesticides, in addition to direct mortality cause sublethal effects, including damage to target selective honey bee organs. The midgut is the organ responsible for the digestion and absorption of nutrients and the detoxification of ingested substances, such as pesticides. This study evaluated the histopathological and cytotoxic changes in the midgut of A. mellifera workers caused by the pesticide azoxystrobin. The limit-test was performed, and a 100 µg a.i./bee dose was administered orally and midgut analyzed with light and transmission electron microscopies after 24 h and 48 h of pesticide exposure. The midgut of the control bees has a single layer of digestive cells, with spherical nuclei, nests of regenerative cells, and the lumen coated with the peritrophic matrix. The bees fed on azoxystrobin showed morphological changes, including intense cytoplasm vacuolization and cell fragments released into the gut lumen. The protein detection test showed greater staining intensity in the nests of regenerative cells after 24 h of exposure to azoxystrobin. The occurrence of damage to the midgut in A. mellifera exposed to azoxystrobin indicates that although this fungicide has been classified as low toxicity for bees, it has sublethal effects in the midgut, and effects in other organs should be investigated.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aki T, Nara A, Uemura K (2012) Cytoplasmic vacuolization during exposure to drugs and other substances. Cell Biol Toxicol 28:125–131. https://doi.org/10.1007/s10565-012-9212-3
Antúnez K, Invernizzi C, Mendoza Y, vanEngelsdorp D, Zunino P (2017) Honey bee colony losses in Uruguay during 2013–2014. Apidologie 48:364–370. https://doi.org/10.1007/s13592-016-0482-2
Arnoult D, Carneiro L, Tattoli I, Girardin SE (2009) The role of mitochondria in cellular defense against microbial infection. Semin Immunol 21:223–232. https://doi.org/10.1016/j.smim.2009.05.009
Aumuller G, Wilhelm B, Seitz J (1999) Apocrine secretion - Fact or artifact? Ann Anat 181:437–446. https://doi.org/10.1016/s0940-9602(99)80020-x
Bancroft JD, Gamble M (2008) Theory and practice of histological techniques. Churchill Livingstone, London
Bartlett DW, Clough JM, Godwin JR, Hall AA, Hamer M, Parr-Dobrzanski B (2002) The strobilurin fungicides. Pest Manage Sci 58:649–662. https://doi.org/10.1002/ps.520
Batista AC, Domingues CED, Costa MJ, Silva-Zacarin ECM (2020) Is a strobilurin fungicide capable of inducing histopathological effects on the midgut and Malpighian tubules of honey bees? J Apicult Res 59: 834-843. https://doi.org/10.1080/00218839.2020.1724678
Böhme F, Bischoff G, Zebitz CPW, Rosenkranz P, Wallner K (2017) Chronic exposure of honeybees, Apis mellifera (Hymenoptera: Apidae), to a pesticide mixture in realistic field exposure rates. Apidologie 48:353–363. https://doi.org/10.1007/s13592-016-0479-x
Brodschneider R, Gray A, van der Zee R, Adjlane N, Brusbardis V, Charrière JD, Woehl S (2016) Preliminary analysis of loss rates of honey bee colonies during winter 2015/16 from the COLOSS survey. J Apic Res 55:375–378. https://doi.org/10.1080/00218839.2016.1260240
Campbell JB, Nath R, Gadau J, Fox T, DeGrandi-Hoffman G, Harrison JF (2016) The fungicide Pristine® inhibits mitochondrial function in vitro but not flight metabolic rates in honey bees. J Insect Physiol 86:11–16. https://doi.org/10.1016/j.jinsphys.2015.12.003
Carneiro LS, Martínez LC, Gonçalves WG, Santana LM, Serrão JE (2019) The fungicide iprodione affects midgut cells of non-target honey bee Apis mellifera workers. Ecotox Environ Saf 189:109991. https://doi.org/10.1016/j.ecoenv.2019.109991
Carneiro LS, Martínez LC, Oliveira AH, Cossolin JFS, Resende MTCS, Gonçalves WG, Medeiros-Santana L, Serrão JE (2022) Acute oral exposure to imidacloprid induces apoptosis and autophagy in the midgut of honey bee Apis mellifera workers. Sci Total Environ 815:152847. https://doi.org/10.1016/j.scitotenv.2021.152847
Castilhos D, Bergamo GC, Gramacho KP, Goncalves LS (2019) Bee colony losses in Brazil: a 5-year online survey. Apidologie 50:263–272. https://doi.org/10.1007/s13592-019-00642-7
Catae AF, Roat TC, De Oliveira RA, Ferreira Nocelli RC, Malaspina O (2014) Cytotoxic effects of thiamethoxam in the midgut and malpighian tubules of Africanized Apis mellifera (Hymenoptera: Apidae). Micros Res Techniq 77:274–281. https://doi.org/10.1002/jemt.22339
Chauzat MP, Bougeard S, Hendrikx P, Ribière-Chabert M (2016) Risk indicators affecting honeybee colony survival in Europe: one year of surveillance. Apidologie 47:348–378. https://doi.org/10.1007/s13592-016-0440-z
Christen V, Krebs J, Fent K (2019) Fungicides chlorothanolin, azoxystrobin and folpet induce transcriptional alterations in genes encoding enzymes involved in oxidative phosphorylation and metabolism in honey bees (Apis mellifera) at sublethal concentrations. J Hazard Mater 377:215–226. https://doi.org/10.1016/j.jhazmat.2019.05.056
Cruz ADS, Silva-Zacarin ECM, da, Bueno OC, Malaspina O (2010) Morphological alterations induced by boric acid and fipronil in the midgut of worker honeybee (Apis mellifera L.) larvae: morphological alterations in the midgut of A. mellifera. Cell Biol Toxicol 26:165–176. https://doi.org/10.1007/s10565-009-9126-x
Dai P, Jack CJ, Mortensen AN, Bloomquist JR, Ellis JD (2018) The impacts of chlorothalonil and diflubenzuron on Apis mellifera L. larvae reared in vitro. Ecotox Environ Saf 164:283–288. https://doi.org/10.1016/j.ecoenv.2018.08.039
Decourtye A, Devillers J, Genecque E, Le Menach K, Budzinski H (2005) Comparative sublethal toxicity of nine pesticides on olfactory learning performances of the honeybee Apis mellifera. Arch Environ Contam Toxicol 48:242–250. https://doi.org/10.1007/s00244-003-0262-7
Decourtye A, Lacassie E, Pham-Delégue MH (2003) Learning performances of honeybees (Apis mellifera L) are differentially affected by imidacloprid according to the season. Pest Manage Sci 59:269–278. https://doi.org/10.1002/ps.631
Denecke S, Swers L, Douris V, Vontas J (2018) How do oral insecticidal compounds cross the insect midgut epithelium? Insect Biochem Mol Biol 103:22–35. https://doi.org/10.1016/j.ibmb.2018.10.005
Desneux N, Decourtye A, Delpuech JM (2007) The sublethal effects of pesticides on beneficial arthropods. Annu Rev Entomol 52:81–106. https://doi.org/10.1146/annurev.ento.52.110405.091440
Devillers J, Pham-Delègue MH (2002) Honey bees: estimating the environmental impact of chemicals. Taylor & Francis, Londres
Domingues CED, Inoue LVB, Silva-Zacarin ECM, Malaspina O (2020a) Fungicide pyraclostrobin affects midgut morphophysiology and reduces survival of Brazilian native stingless bee Melipona scutellaris. Ecotoxicol Environ Saf 206. https://doi.org/10.1016/j.ecoenv.2020.111395
Domingues CED, Inoue LVB, Silva-Zacarin ECM, Malaspina O (2020b) Foragers of Africanized honeybee are more sensitive to fungicide pyraclostrobin than newly emerged bees. Environ Pollut 266:115267. https://doi.org/10.1016/j.envpol.2020.115267
Doonan F, Cotter TG (2008) Morphological assessment of apoptosis. Methods 44:200–204. https://doi.org/10.1016/j.ymeth.2007.11.006
EFSA (2010) Conclusion on the peer review of the pesticide risk assessment of the active substance azoxystrobin. EFSA Journal 8:1542. https://doi.org/10.2903/j.efsa.2010.1542
Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35:495–516. https://doi.org/10.1080/01926230701320337
EPA (2008) Pesticide Fact Sheet Name of Chemical: Azoxystrobin. {http: //www.epa.gov/opp00001/chem_search/reg_actions/registration/fs_PC-090100_01-Apr-08.pdf} Accessed November 2019.
Farder-Gomes CF, Fernandes KM, Bernardes RC, Bastos DSS, Martins GF, Serrão JE (2021) Acute exposure to fipronil induces oxidative stress, apoptosis and impairs epithelial homeostasis in the midgut of the stingless bee Partamona helleri Friese (Hymenoptera: Apidae). Sci Total Environ 774:145679. https://doi.org/10.1016/j.scitotenv.2021.152847
Ferreira C, Bellinello GL, Ribeiro AF, Terra WR (1990) Digestive enzymes associated with the glycocalyx, microvillar membranes and secretory vesicles from midgut cells of Tenebrio molitor larvae. Insect Biochem 20:839–847
Fiaz M, Martínez LC, Plata-Rueda A, Gonçalves WG, de Souza DLL, Cossolin JFS, Carvalho P, Martins G, Serrão JE (2019) Pyriproxyfen, a juvenile hormone analog, damages midgut cells and interferes with behaviors of Aedes aegypti larvae. PeerJ 7:e7489. https://doi.org/10.7717/peerj.7489
Fisher A, DeGrandi-Hoffman G, Smith BH, Johnson M, Kaftanoglu O, Cogley T, Fewell JH, Harrison JF (2021) Colony field test reveals dramatically higher toxicity of a widely-used mito-toxic fungicide on honey bees (Apis mellifera). Environ Pollut 269:115964. https://doi.org/10.1016/j.envpol.2020.115964
Forkpah C, Dixon LR, Fahrbach SE, Rueppell O (2014) Xenobiotic effects on intestinal stem cell proliferation in adult honey bee (Apis mellifera L) workers. PLoS One 9:e91180. https://doi.org/10.1371/journal.pone.0091180
Han W, Yang Y, Gao J, Zhao D, Ren C, Wang S, Zhong Y (2019) Chronic toxicity and biochemical response of Apis cerana cerana (Hymenoptera: Apidae) exposed to acetamiprid and propiconazole alone or combined. Ecotoxicology 28:399–411. https://doi.org/10.1007/s10646-019-02030-4
Hladik ML, Vandever M, Smalling KL (2016) Exposure of native bees foraging in an agricultural landscape to current-use pesticides. Sci Total Environ 542:469–477. https://doi.org/10.1016/j.scitotenv.2015.10.077
Iwasaki JM, Hogendoorn K (2021) Non-insecticide pesticide impacts on bees: A review of methods and reported outcomes. Agric Ecosyst Environ 314:107423. https://doi.org/10.1016/j.agee.2021.107423
Johnson RM, Dahlgren L, Siegfried BD, Ellis MD (2013) Acaricide, fungicide and drug interactions in honey bees (Apis mellifera). PloS One 8:e54092. https://doi.org/10.1371/journal.pone.0054092
Kakamand FAK, Mahmoud TT, Amin ABM (2008) The role of three insecticides in disturbance the midgut tissue in honey bee Apis mellifera L. workers. J Dohuk Univ 11:144–151
Kim JH, Campbell BC, Mahoney N, Chan KL, Molyneux RJ, May GS (2007) Enhanced activity of strobilurin and fludioxonil by using berberine and phenolic compounds to target fungal antioxidative stress response. Lett Appl Microbiol 45:134–141. https://doi.org/10.1111/j.1472-765X.2007.02159.x
Klein AM, Vaissière BE, Cane JH, Steffan-Dewenter I, Cunningham SA, Kremen C, Tscharntke T (2007) Importance of pollinators in changing landscapes for world crops. Proc R Soc B Biol Sci 274:303–313. https://doi.org/10.1098/rspb.2006.3721
Köhler HR, Triebskorn R (1998) Assessment of the cytotoxic impact of heavy metals on soil invertebrates using a protocol integrating qualitative and quantitative components. Biomarkers 3:109–127. https://doi.org/10.1080/135475098231273
Leroux P (1996) Recent developments in the mode of action of fungicides. Pestic Sci 47:191–197
Loiseleur O (2017) Natural products in the discovery of agrochemicals. CHIMIA Int J Chem 71:810–822. 0.2533/chimia.2017.810
Lopes MP, Fernandes KM, Ventura-Tome HV, Goncalves WG, Miranda FR, Serrão JE, Martins GF (2018) Spinosad-mediated effects on the walking ability, midgut, and Malpighian tubules of Africanized honey bee workers. Pest Manage Sci 74:1311–1318. https://doi.org/10.1002/ps.4815
Martínez LC, Plata-Rueda A, Gonçalves WG, Freire AFPA, Zanuncio JC, Bozdoğan H, Serrão JE (2019) Toxicity and cytotoxicity of the insecticide imidacloprid in the midgut of the predatory bug, Podisus nigrispinus. Ecotox Environ Saf 167:69–75. https://doi.org/10.1016/j.ecoenv.2018.09.124
Martínez LC, Plata-Rueda A, Neves G, da S, Gonçalves WG, Zanuncio JC, Bozdoğan H, Serrão JE (2018) Permethrin induces histological and cytological changes in the midgut of the predatory bug, Podisus nigrispinus. Chemosphere 212:629–637. https://doi.org/10.1016/j.chemosphere.2018.08.134
Medrzycki P, Giffard H, Aupinel P, Belzunces LP, Chauzat M et al. (2013) Standard methods for toxicology research in Apis mellifera. J Apic Res 52:1–60. https://doi.org/10.3896/IBRA.1.52.4.14
Michener CD (1974) The social behavior of the bees: A comparative study. Havard Univiversity Press, Cambridge.
Moraes RLS, Bowen ID (2000) Modes of cell death in the hypopharyngeal gland of the honey bee (Apis mellifera L.). Cell Biol Int 24:737–743. https://doi.org/10.1006/cbir.2000.0534
OECD (Organization for Economic Co-operation and Development). (1998) OECD - guidelines for the testing of chemicals, honeybees. Acute Oral ToxicityTest n°213.
Oliveira RA, Roat TC, Caravalho SM, Malaspina O (2012) Side-effects of thiamethoxam on the brain and midgut of the africanizaed honeybee Apis mellifera (Hymenoptera: Apidae). Environ Toxicol 29:1122–1133. https://doi.org/10.1002/tox.21842
O’Neal ST, Reeves AM, Fell RD, Brewster CC, Anderson TD (2019) Chlorothalonil exposure alters virus susceptibility and markers of immunity, nutrition, and development in honey bees. J Insect Sci 19:14. https://doi.org/10.1093/jisesa/iez051
Ostiguy N, Drummond FA, Aronstein K, Eitzer B, Ellis JD, Spivak M, Sheppard WS (2019) Honey bee exposure to pesticides: a four-year nationwide study. Insects 10:13. https://doi.org/10.3390/insects10010013
Piechowicz B, Sadło S, Woś I, Białek J, Depciuch J, Podbielska M, Szpyrka E, Kazioł K, Piechowicz I, Koziorowska A (2020) Treating honey bees with an extremely low frequency electromagnetic field and pesticides: Impact on the rate of disappearance of azoxystrobin and λ-cyhalothrin and the structure of some functional groups of the probabilistic molecules. Environ Res 190:109989. https://doi.org/10.1016/j.envres.2020.109989
Proskuryakov SY, Konoplyannikov AG, Gabai VL (2003) Necrosis: a specific form of programmed cell death. Exp Cell Res 283:1–16. https://doi.org/10.1016/s0014-4827(02)00027-7
Ratnieks FLW, Carreck NL (2010) Clarity on honey bee collapse. Science 327:152–153. https://doi.org/10.1126/science.1185563
Reynolds ES (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 17:208–212
Rossi CA, Roat TC, Tavares DA, Cintra-Socolowski P, Malaspina O (2013) Effects of sublethal doses of imidacloprid in malpighian tubules of africanized Apis mellifera (Hymenoptera, Apidae). Micros Res Techniq 76:552–558. https://doi.org/10.1002/jemt.22199
Santos-Junior VC, dos, Martínez LC, Plata-Rueda A, Fernandes FL, Tavares W, de S, Zanuncio JC, Serrão JE (2020) Histopathological and cytotoxic changes induced by spinosad on midgut cells of the non-target predator Podisus nigrispinus Dallas (Heteroptera: Pentatomidae). Chemosphere 238:124585. https://doi.org/10.1016/j.chemosphere.2019.124585
Schwarz JM, Knauer AC, Allan MJ, Dean RR, Ghazoul J, Tamburini G, Wintermantel D, Klein AM, Albrecht M (2022) No evidence for impaired solitary bee fitness following pre-flowering sulfoxaflor application alone or in combination with a common fungicide in a semi-field experiment. Environ Int 164:107252. https://doi.org/10.1016/j.envint.2022.107252
Serra RS, Cossolin JFS, Resende MTCS, Arthidoro de Castro M, Oliveira AH, Martinez LC, Serrão JE (2021) Spiromesifen induces histopathological and cytotoxic changes in the midgut of the honeybee Apis mellifera (Hymenoptera: Apidae). Chemosphere 270:129439. https://doi.org/10.1016/j.chemosphere.2020.129439
Serrão JE, Plata-Rueda A, Martínez LC, Zanuncio JC (2022) Side-effects of pesticides on non-target insects in agriculture: a mini-review. Sci Nat 109:17. https://doi.org/10.1007/s00114-022-01788-8
Simon-Delso N, San Martin G, Bruneau E, Hautier L, Medrzycki P (2017) Toxicity assessment on honey bee larvae of a repeated exposition of a systemic fungicide, boscalid. Bull Insectol 70:83–89
Sponsler DB, Grozinger CM, Hitaj C, Rundlöf M, Botías C, Code A, Lonsdorf EV, Melathopoulos AP, Smith DJ, Suryanarayanan S, Thogmartin WE, Williams NM, Zhang M, Douglas MR (2019) Pesticides and pollinators: A socioecological synthesis. Sci Total Environ 662:1012–1027. https://doi.org/10.1016/j.scitotenv.2019.01.016
Stefanini M, Demartino C, Zamboni L (1967) Fixation of ejaculated spermatozoa for electron microscopy. Nature 216:173–174
Steinmann N, Corona M, Neumann P, Dainat B (2015) Overwintering is associated with reduced expression of immune genes and higher susceptibility to virus infection in honey bees. PLoS One 10:e0129956. https://doi.org/10.1371/journal.pone.0129956
Stoner KA, Cowles RS, Nurse A, Eitzer BD (2019) Tracking pesticide residues to a plant genus using palynology in pollen trapped from honey bees (Hymenoptera: Apidae) at ornamental plant nurseries. Environ Entomol 48:351–362. https://doi.org/10.1093/ee/nvz007
Straw EA, Brown MJF (2021) Co-formulant in a commercial fungicide product causes lethal and sub-lethal effects in bumble bees. Sci Rep 11:21653. https://doi.org/10.1038/s41598-021-00919-x.
Tadei R, Menezes-Oliveira VB, Silva-Zacarin ECM (2020) Silent effect of the fungicide pyraclostrobin on the larval exposure of the non-target organism Africanized Apis mellifera and its interaction with the pathogen Nosema ceranae in adulthood. Environ Pollut 267:115622. https://doi.org/10.1016/j.envpol.2020.115622
Tamburini G, Wintermantel D, Allan MJ, Dean RR, Knauer A, Albrecht M, Klein AM (2021a) Sulfoxaflor insecticide and azoxystrobin fungicide have no major impact on honeybees in a realistic-exposure semi-field experiment. Sci Total Environ 778:146084. https://doi.org/10.1016/j.scitotenv.2021.146084
Tamburini G, Pereira-Peixoto MH, Borth J, Lotz S, Wintermantel D, Allan MJ, Dean R, Schwarz JM, Knauer A, Albrecht M, Klein AM (2021b) Fungicide and insecticide exposure adversely impacts bumblebees and pollination services under semi-field conditions. Environ Int 157:106813. https://doi.org/10.1016/j.envint.2021.106813
Williams GR, Alaux C, Costa C, Csaki T, Doublet V, Eisenhardt D, Fries I, Kuhn R, McMahon DP, Medrzycki P, Murray TE, Natsopoulou ME, Neumann P, Oliver R, Paxton RJ, Pernal SF, Shutler D, Tanner G, van der Steen JJM, Brodschneider R (2013) Standard methods for maintaining adult Apis mellifera in cages under in vitro laboratory conditions. J Apicult Res 52:52104. https://doi.org/10.3896/ibra.1.52.1.04
Wu T, Han B, Wang X, Tong Y, Liu F, Diao Q, Dai P (2022) Chlorothalonil alters the gut microbiota and reduces the survival of immature honey bees reared in vitro. Pest Manage Sci 78:1976–1981. https://doi.org/10.1002/ps.6816
Zaluski R, Justulin LA, Orsi RD (2017) Field-relevant doses of the systemic insecticide fipronil and fungicide pyraclostrobin impair mandibular and hypopharyngeal glands in nurse honeybees (Apis mellifera). Sci Rep 7:15217. https://doi.org/10.1038/s41598-017-15581-5
Zhivotovsky B (2004) Apoptosis, necrosis and between. Cell Cycle 3:64–66. https://doi.org/10.4161/cc.3.1.606
Zioga E, Kelly R, White B, Stout JC (2020) Plant protection product residues in plant pollen and nectar: A review of current knowledge. Environ Res 189:109873. https://doi.org/10.1016/j.envres.2020.109873
Acknowledgements
The authors are grateful to Nucleus of Microscopy and Microanalysis of the Universidade Federal de Viçosa for technical assistance.
Funding
This research was supported by Brazilian research agencies CAPES (code 001), CNPq (303467/2018-5), and FAPEMIG (02367-18).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Serra, R.S., Martínez, L.C., Cossolin, J.F.S. et al. The fungicide azoxystrobin causes histopathological and cytotoxic changes in the midgut of the honey bee Apis mellifera (Hymenoptera: Apidae). Ecotoxicology 32, 234–242 (2023). https://doi.org/10.1007/s10646-023-02633-y
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10646-023-02633-y