Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Biochemical and histological biomarkers in the midgut of Apis mellifera from polluted environment at Beheira Governorate, Egypt

  • 390 Accesses

  • 6 Citations


The aim of this study was to analyze the impact of organophosphorus (OP) pollutants on oxidative stress and ultrastructural biomarkers in the midgut of the honeybee Apis mellifera collected from three locations that differ in their extent of spraying load with OP insecticides: a weakly anthropised rural site, Bolin which is considered as a reference site; moderately spraying site, El Kaza; and a strongly anthropised urban site, Tiba with a long history of pesticide use. Results showed that high concentrations of chlorpyrifos, malathion, diazinon, chlorpyrifos-methyl, and pirimiphos-methyl were detected in midgut at locations with extensive pesticide spraying. Reduced glutathione content, superoxide dismutase, catalase, and glutathione peroxidase displayed lowest activities in the heavily sprayed location (Tiba). Lipid peroxidation level in the midgut of honeybees in the sprayed locations was found to be significantly higher compared to the reference values. Meanwhile, various ultrastructural abnormalities were observed in the epithelial cells of midgut of honeybees collected from El Kaza and Tiba, included confluent and disorganized microvilli and destruction of their brush border, the cytoplasm with large vacuoles and alteration of cytoplasmic organelles including the presence of swollen mitochondria with lysis of matrices, disruption of limiting membranes, and disintegration of cristae. The nuclei with indented nuclear envelope and disorganized chromatin were observed. These investigated biomarkers indicated that the surveyed honeybees are being under stressful environmental conditions. So, we suggest using those biomarkers in the assessment of environmental quality using honeybees in future monitoring of ecotoxicological studies.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. Abdel-Aziz AA (2006) The use of chemigation technique for weed control and minimizing environmental pollution with herbicides in newly cultivated lands. Misr J Ag Eng 23(3):571–592

  2. Abd El-Aziz HS, Hanan S, Mansy MS, Desuky WM, Elyassaki SM (2007) Toxicity evaluation and biochemical impacts of some Bacillus thuringiensis formulation on Spodoptera littoralis larvae. J Egypt Acd Soc Environ Develop 8:1–10

  3. Abdel-Halim KY, Abu El-Saad AM, Talha MM, Hussein AA, Bakry NM (2013) Oxidative stress on land snail Helix aspersa as a sentinel organism for ecotoxicological effects of urban pollution with heavy metals. Chemosphere 93(6):1131–1138

  4. Abdel-Moneim AM, Essawy A, Badr El-Din NK, El-Naggar NM (2013) Biochemical and histopathological changes in liver of the Nile tilapia from Egyptian polluted lakes. Toxicol Ind Health 5:1–11

  5. Abdelsalam SA, Alzahrani AM, Elmenshawy OM, Abdel-Moneim AM (2016) Spinosad induces antioxidative response and ultrastructure changes in males of red palm weevil Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae). J Ins Sci 16(1):1–10

  6. Abdollahi M, Ranjbar A, Shadnia S, Nikfar S, Rezaie A (2004) Pesticides and oxidative stress: a review. Med Sci Monit 10:141–147

  7. Abhilash PC, Singh N (2009) Pesticide use and application: an Indian scenario. J Hazard Mater 165:1–12

  8. Abu El-Saad AM (2010) Biochemical markers of oxidative stress in the terrestrial isopod, Porcellio leavis exposed to environmental contamination at different regions of Alexandria, Egypt. Proc 6th Int Con Biol Sci (Zool) 6:202–2015

  9. Abu El-Saad AM, Elgerbed MS (2010) Dimethoate-induced hepatotoxicity in rats and the roles of vitamin E and N-acetylcysteine. Egypt J Exp Biol (Zoo) 6:219–230

  10. Adel MM, Sammour EA (2012) Effect of sub-lethal dose of natural compound of Medicago sativa (L, Leguminaceae) on the hind gut and fat body of Spodoptera littoralis (Lepidoptera, Noctuidae). J Appl Sci Res 8:1398–1408

  11. Adel MM, El-Hawary FM, Abdel-Aziz NF, Sammour EA (2010) Some physiological, biochemical and histopathological effects of Artemisia monosperma against the cotton leafworm, Spodoptera littoralis. Arch Phytopathol Plant Prot 43:1098–1110

  12. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126

  13. Ahmad S (1992) Biochemical defence of pro-oxidant plant allelochemicals by herbivorous insects. Biochem Syst Ecol 20:269–296

  14. Akbar SMD, Sharma HC, Jayalakshmi SK, Sreeramulu K (2012) Effect of pyrethroids, permethrin and fenvalarate, on the oxidative stress of Helicoverpa armigera. World J Sci Technol 2:01–05

  15. Alaux C, Brunet JL, Dussaubat C, Mondet F, Tchamitchan S, Cousin M, Brillard J, Baldy A, Belzunces LP, LeConte Y (2010) Interactions between Nosema microspores and a neonicotinoid weaken honeybees (Apis mellifera). Environ Microbiol 12:774–782

  16. Ambika A, Selvisabhanayakam (2012) Toxicity studies on the effect of latex on the adult male insect, Odontopus varicornis (Heteroptera: Pyrrhocoridae). Intern J Toxicol Appl Pharmacol 2249–9709

  17. Amr MM (1999) Pesticide monitoring and its health problems in Egypt, a third world country. Toxicol Lett 107:1–13

  18. Anderson ME (1989) Enzymatic and chemical methods for the determination of glutathione. In: Dolphin D, Poulson R, Avramovic O (eds) Glutathione: chemical, biochemical and medical aspects. New York, NY, John Wiley and Sons pp, pp. 339–365

  19. Aslanturk A, Kalender S, Uzunhisarcikli M, Kalender Y (2011) Effects of methidathion on antioxidant enzyme activities and malondialdehyde level in midgut tissues of Lymantria dispar (Lepidoptera) larvae. J Entomol Res Soc 13:27–38

  20. Au DWT (2004) The application of histo-cytopathological biomarkers in marine pollution monitoring: a review. Mar Pollut Bull 48:817–834

  21. Augustyniak M, Babczynska A, Migula P, Wilczek G, Laszczyca P, Kafel AM, Augustyniak M (2005) Joint effects of dimethoate and heavy metals on metabolic responses in a grasshopper (Chorthippus brunneus) from a heavy metals pollution gradient. Comp Biochem Physiol 141:412–419

  22. Augustyniak M, Babczyńska A, Augustyniak M (2009) Does the grasshopper Chorthippus brunneus adapt to metal polluted habitats? A study of glutathione-dependent enzymes in grasshopper nymphs. Insect Sci 16:33–42

  23. Badawy M, Nasr H, Rabea E (2015) Toxicity and biochemical changes in the honey bee Apis mellifera exposed to four insecticides under laboratory conditions. Apidologie 46:177–193

  24. Banerjee BD, Seth V, Bhattacharya A, Pasha ST, Chakraborty AK (1999) Biochemical effects of some pesticides on lipid peroxidation and free radical scavangers. Toxicol Lett 107:33–47

  25. Ben Amara I, Soudani N, Troudi A, Bouaziz H, Boudawara T, Zeghal N (2011) Antioxidant effect of vitamin E and selenium on hepatotoxicity induced by dimethoate in female adult rats. Ecotoxicol Environ Saf 74:811–819

  26. Bénéteau B, Carvalho SM, Brunet JL, Carvalho GA, Buleté A, Giroud B, Belzunces LP (2012) Development of biomarkers of exposure to xenobiotics in the honey bee Apis mellifera: application to the systemic insecticide thiamethoxam. Ecotoxicol Environ Saf 82:22–31

  27. Bénéteau B, Benneveau A, Geret F, Delatte H, Becker N, Belzunces (2013) Honeybee biomarkers as promising tools to monitor environmental quality.Environ Int 60:31–41

  28. Billingsley PF, Lehane MJ (1996) Structure and ultrastructure of the insect midgut. In: Lehane MJ, Billingsley PF (eds) Biology of the insect midgut. Chapman and Hall, London, pp. 3–30 486p

  29. Bizhannia AR, Etebari K, Sorati R (2005) The effects of juvenile hormone analogue, admiral, application on protein metabolism of silkworm larvae. J Entomol Soc Iran 25:43–56

  30. Boily B, DeBlois C, Aras P, Chagnon M (2013) Acetylcholinesterase in honey bees (Apis mellifera) exposed to neonicotinoids, atrazine and glyphosate: laboratory and field experiments. Environ Sci Pollut Res 20:5603–5614

  31. Braeckman B, Smagghe G, Brutsaert N, Cornelis R, Raes H (1999) Cadmium uptake and defense mechanism in insect cells. Environ Res 80:231–243

  32. Bream AS (2003) Laboratory evaluation of heavy metals stress on certain biochemical parameters of the aquatic insect, Sphaerodema urinator Duf. (Hemiptera: Belostomatidae). Commun Agric Appl Biol Sci 68:291–297

  33. Carvalho SM, Belzunces LP, Carvalho GA, Brunet JL, Beneteau B (2013) Enzymatic biomarkers as tools to assess environmental quality: a case study of exposure of the honey bee Apis mellifera to insecticides. Environ Toxicol Chem 32:2117–2124

  34. Cavados C, Majerowicz S, Chaves JQ, Rabinovitch LL (2004) Histopathological and ultrastructural effects of δ-endotoxins of Bacillus thuringiensis serovar israelensis in the midgut of Simulium pertinax larvae (Diptera, Simulidae). Mem Inst Oswaldo Cruz 99:493–498

  35. Chakrabarti P, Rana S, Smith B, Basu P (2015) Pesticide-induced oxidative stress in laboratory and field populations of native honey bees along intensive agricultural landscapes in two Eastern Indian states. Apidologie 46:107–129

  36. Chandran R, Sivakumar A, Mohandass S, Aruchami M (2005) Effect of cadmium and zinc on antioxidant enzyme activity in the gastropod, Achatinafulica. Comp Biochem Physiol 140:422–426

  37. Chauzat MP, Carpentier P, Martel AC, Bougeard S, Cougoule N, Porta P (2009) Influence of pesticides residues in honeybee (Hymenoptera: Apidae) colony health in France. Environ Entomol 38:514

  38. Chu FF, Doroshow JH, Esworthy RS (1993) Expression, characterization, and tissue distribution of a new cellular selenium dependent glutathione peroxidase, GSHPx-GI. J Biol Chem 268:2571–2576

  39. Codling G, Naggar Y, Giesy J, Robertson A (2016) Concentrations of neonicotinoid insecticides in honey, pollen and honey bees (Apis mellifera L.) in central Saskatchewan, Canada. Chemosphere 144:2321–2328

  40. Cristofoletti PT, Ribeiro AF, Terra WR (2001) Apocrine secretion of amylase and exocytosis of trypsin along the midgut of Tenebrio molitor larvae. J Insect Physiol 47:143–155

  41. Dallinger R (1993) Strategies of metal detoxification in terrestrial invertebrates. In: Dallinger R, Rainbow PS (eds) Ecotoxicology of metals in invertebrates. Lewis Publishers, Boca Raton ISBN 0873717341, UK, pp. 246–289

  42. Damiens G, Gnassia-Barelli M, Loquès F, Roméo M, Salbert V (2007) Integrated biomarker response index as a useful tool for environmental assessment evaluated using transplanted mussels. Chemosphere 66:574–583

  43. Decio P, Silva-Zacarin ECM, Bueno FC, Bueno OC (2013) Toxicological and histopathological effects of hydramethylnon on Atta sexdens rubropilosa (Hymenoptera: Formicidae) workers. Micron 45:22–31

  44. Diaz AN, Parwja AG, Sanchez FG (1997) Liquid and gas chromatographic multi-residue pesticide determination in animal tissues. Pestic Sci 49:56–64

  45. Dickinson DA, Forman HJ (2002) Cellular glutathione and thiols metabolism. Biochem Pharmacol 64:1019–1026

  46. Dimitrova MS, Tsinova V, Velcheva V (1994) Combined effect of zinc and lead on the hepatic superoxide dismutase–catalase system in carp (Cyprinus carpio). Comp Biochem Physiol 108:43–46

  47. El-barky NM, Dahi HF, El-Sayed YA (2008) Toxicological evaluation and biochemical impacts for radient as a new generation of spinosyn of Spodoptera littoralis (Boisd.), larvae. Egypt. Acad J Biolog Sci 1:85–97

  48. Fairbrother A, Purdy J, Anderson T, Fellk R (2014) Risks of neonicotinoid insecticides to honey bees. Environ Toxicol Chem 33(4):719–731

  49. Felton GW, Summers CB (1995) Antioxidant systems in insects. Arch Insect Biochem Physiol 29:187–197

  50. Fernandes D, Porte C, Bebianno MJ (2007) Chemical residues and biochemical responses in wild and cultured European sea bass (Dicentrarchus labrax L.). Environ Res 103:247–256

  51. Gauthier M, Aras P, Jumarie C, Boily M (2016) Low dietary levels of Al, Pb and Cd may affect the non-enzymatic antioxidant capacity in caged honey bees (Apis mellifera). Chemosphere 144:848–854

  52. Gultekin F, Ozturk M, Akdogan M (2000) The effect of organophosphate insecticide chlorpyrifos-ethyl on lipid peroxidation and antioxidant enzymes (in vitro). Arch Toxicol 74:533–538

  53. Hardstone MC, Scott JG (2010) Is Apis mellifera more sensitive to insecticides than other insects. Pest Manag Sci 66(11):1171–1180

  54. Henry M, Beguin M, Requier F, Rollin O, Odoux JF, Aupinel P (2012) A common pesticide decreases foraging success and survival in honey bees. Sci 336:348–350

  55. 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

  56. Kalender S, Kalender Y, Durak D, Ogutcu A, Uzunhisarcikli M, Cevrimli BS, Yildirim M (2007) Methyl parathion induced nephrotoxicity in male rats and protective role of vitamins C and E. Pestic Biochem Phys 88:213–218

  57. Khaled R (2014) Agricultural pesticidesillegal pesticides threaten agriculture and fragile ecosystem. Egypt Independent’s weekly print edition 11:39

  58. Khan I, Qamar A, Mehdi SH, Shahid M (2011) Histopathological effects of Datura alba leaf extract on the midgut of Periplaneta americana. Biol Med 3:260–264

  59. Krishnan N, Kodrik DO (2006) Antioxidant enzymes in Spodoptera littoralis (Boisduval): are they enhanced to protect gut tissues during oxidative stress? J Insect Physiol 52:11–20

  60. Krishnan N, Kodrík D, Kłudkiewicz B, Sehnal F (2009) Glutathione-ascorbic acid redox cycle and thioredoxin reductase activity in the digestive tract of Leptinotarsa decemlineata (Say). Insect Biochem Mol Biol 39:180–188

  61. Krupke CH, Hunt GJ, Eitzer BD, Andino G, Given K (2012) Multiple routes of pesticide exposure for honey bees living near agricultural fields. PLoS One 7:29–48

  62. Lambert O, Veyrand B, Durand S, Marchand P, Bizec B, Piroux M, Puyo S, Thorin C, Frédéric D (2012) Polycyclic aromatic hydrocarbons: bees, honey and pollen as sentinels for environmental chemical contaminants. Chemosphere 86:98–104

  63. Linde-Arias AR, Inacio AF, Novo LA, Alburquerque C, Moreira JC (2008) Multibiomarker approach in fish to assess the impact of pollution in a large Brazilian river, Paraiba do Sul. Environ Poll 156:974–979

  64. Lionetto MG, Caricato R, Giordano ME, Pascariello MF, Marinosci L, Schettino T (2003) Integrated use of biomarkers (acetylcholinesterase and antioxidant enzymes activities) in Mytilus galloprovincialis and Mullus barbatus in an Italian coastal marine area. Mar Pollut Bull 46:324–330

  65. Liu TP (1984) Ultrastructure of the midgut of the worker honey Apis mellifera heavily infected with Nosema apis. J Invertebr Pathol 44:282–291

  66. Lowry OH, Rasebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275

  67. Mamidala P, Jones S, Mittapalli O (2011) Metabolic resistance in bed bugs. Insects 2(1):36–48

  68. Mansour SA (2004) Pesticides exposure—Egyptian scene. Toxicol 198:91–115

  69. Mansour S, Mossa A (2009) Lipid peroxidation and oxidative stress in rat erythrocytes induced by chlorpyrifos and the protective effect of zinc. Pestic Biochem Physiol 97:34–39

  70. Meyer JN, Maxwell CKL, Rooney JP, Michael ASO, Kisby HE, Bess AS (2013) Mitochondria as a target of environmental toxicants. Toxicol Sci 134:1–17

  71. Nair V, Turner GE (1984) The thiobarbituric acid test for lipid peroxidation structure of the adduct with malondialdehyde. Lipids 19:84–95

  72. Nebot C, Moutet M, Huet P (1993) Spectrophotometric assay of superoxide dismutase activity based on the activated autoxidation of a tetracyclic catechol. Anal Biochem 214:442–451

  73. Nguyen BK, Saegerman C, Pirard C, Mignon J, Widart J, Tuirionet B, Verheggen FJ, Berkvens D, De Pauw E, Haubruge E (2009) Does imidacloprid seed-treated maize have an impact on honey bee mortality? J Econ Entomol 102:616–623

  74. Osborne JL (2012) Bumble bees and pesticides. Nature 491(7422):43–45

  75. Percy J, Fast PG (1983) Bacillus thuringiensis crystal toxin: ultrastructural studies of its effect on silkworm midgut cells. J Invertebr Pathol 41:86–98

  76. Ragab MM, Antar AS, Talha NI, El-Shahawy MI (2007) Factors affecting chemical DTPA-extractabilily of some heavy metals from different soil types in Egypt. Alex Sci Exch J 28(4):183–190

  77. Ranjbar A, Pasalar P, Abdollahi M (2002) Induction of oxidative stress and acetylcholinesterase inhibition in organophosphorous pesticide manufacturing workers. Hum Exp Toxicol 21:179–182

  78. Ranjini KR, Nambiar JG (2015) Histopathological effects of leaf extracts of Clerodendrum infortunatum and Eupatorium odoratum on the midgut tissue of sixth instar larvae of Orthaga exvinacea Hampson (Lepidoptera: Pyralidae). J Entmol Zool Stud 3:296–301

  79. Rawi SM, Bakry FA, Al-Hazmi MA (2011) Biochemical and histopathological effect of crude extracts on Spodoptera littoralis larvae. J Evol Biol Res 3:67–78

  80. Renzi MT, Amichot M, Pauron D, Tchamitchian S, Brunet J, Maini S (2016) Chronic toxicity and physiological changes induced in the honey bee by the exposure to fipronil and Bacillus thuringiensis spores alone or combined. Ecotoxicol Environ Saf 127:205–213

  81. Roel AR, Dourado DM, Matias R, Porto KR, Bednaski AV, da Costa RB (2010) The effect of sub-lethal doses of Azadirachta indica (Meliaceae) oil on the midgut of Spodoptera frugiperda (Lepidoptera, Noctuidae). Rev Bras entomol 54:0085–5626

  82. Salvemini F, Fraze A, Iervolino A (1999) Enhanced glutathione levels and oxidoresistance mediated by increased glucose-6-phosphate dehydrogenase expression. J Biol Chem 274:2750–2757

  83. Sayed MR, Fayey AB, Al-Hazm MA (2011) Biochemical and histopathological effect of formulated and non-formulated plant extracts on Spodoptera littoralis larvae. Int J Plant Sci 2:107–118

  84. Seidman LA, Bergtrom G, Remsen CC (1986) Structure of the larval midgut of the fly Chironomus thummi and its relationship to sites of cadmium sequestration. Tissue Cell 18:407–418

  85. Schneider CW, Tautz J, Grunewald B, Fuchs S (2012) RFID tracking of sublethal effects of two neonicotinoid insecticides on the foraging behavior of Apis mellifera. PLoS One 7(1):9e30023. doi:10.1371/journal.pone.0030023

  86. Stohs SJ, Bagchi D, Hassoun E, Bagchi M (2000) Oxidative mechanisms in the toxicity of chromium and cadmium ions. J Environ Pathol Toxicol Oncol 19:201–213

  87. Tuzmen N, Candan N, Kays E, Demiryas N (2008) Biochemical effects of chlorpyrifos and deltamethrin on altered antioxidative defense mechanisms and lipid peroxidation in rat liver. Cell Biochem Funct 26:119–124

  88. Vandenbulcke F, Grelle C, Fabre CM, Descamps M (1998) Ultrastructural and autometallographic studies of the nephrocytes of Lithobius forficatus L. (Myriapoda, Chilopoda): role in detoxification of cadmium and lead. Int J Insect Morphol Embryol 27:111–120

  89. Wang P, Wang H, Xu M (2014) Combined subchronic toxicity of dichlorvos with malathion or pirimicarb in mice liver and serum: a metabonomic study. Food Chem Toxicol 70:222–230

  90. Wu C, Ye G, Hu C, Cheng J (2006) Accumulation of cadmium and its effects on growth, development and hemolymph biochemical compositions in Boettcherisca peregrina larvae (Diptera: Sarcophagidae). Insect Sci 13:31–39

  91. Wu H, Liu J, Zhang R, Zhang J, Guo Y, Ma E (2011b) Biochemical effects of acute phoxim administration on antioxidant system and acetyl cholinesterase in Oxya chinensis (Thunberg) (Orthoptera: Acrididae). Pestic Biochem Physiol 100:23–26

  92. Wu JY, Anelli CM, Sheppard WS (2011a) Sub-lethal effects of pesticide residues in brood comb on worker honey bee (Apis mellifera) development and longevity. PLoS One 6:e14720

  93. Zibaee A, Sendi JJ, Etebari K, Alinia F, Ghadamyari M (2008) The effect of diazinon on some biochemical characteristics of Chilo suppressalis Walker (Lepidoptera: Pyralidae), rice striped stem borer. Mun Entomol Zool 3:255–264

Download references

Author information

Correspondence to Ahmed M. Abu El-Saad.

Ethics declarations

Conflict of interest

The authors declared no conflicts of interest.

Additional information

Responsible editor: Philippe Garrigues

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

El-Saad, A.M.A., Kheirallah, D.A. & El-Samad, L.M. Biochemical and histological biomarkers in the midgut of Apis mellifera from polluted environment at Beheira Governorate, Egypt. Environ Sci Pollut Res 24, 3181–3193 (2017). https://doi.org/10.1007/s11356-016-8059-1

Download citation


  • Biomarker
  • Honeybees
  • Biomonitoring
  • Oxidative stress
  • Midgut