Isoenzymes and protein polymorphism in Blaps polycresta and Trachyderma hispida (Forsskål, 1775) (Coleoptera: Tenebrionidae) as biomarkers for ceramic industrial pollution

  • Dalia A. KheirallahEmail author
  • Lamia M. El-Samad


The expression levels of Esterase Isoenzyme and total soluble protein fractionation were studied in two coleopteran insects Blaps polycresta and Trachyderma hispida to evaluate the possible hazards from ceramic and plastic factories in the Khorshed Region, East of Alexandria, Egypt. Two insect collection sites were selected. The first site was the garden of the Faculty of Science, Moharram Bek, Alexandria University, which is considered a non-polluted site, and Khorshed district, considered as the polluted site. Percentages of heavy metals were estimated using SEM-X-ray microanalysis of soft tissues of both sexes of the two coleopteran insects. Esterase Isoenzymes were found to be overexpressed in B. Polycresta but not T. hispida. Female B. polycresta from the polluted site exhibited overexpression of the second and third loci. Furthermore, the females were found to be more affected than males, which only showed the overexpression of the second loci. T. hispida (females and males) collected from the reference site were found to have increased esterase activity compared with those sampled from the polluted site. The Snake-Skin™ Dialysis tubing technique, used for optimizing the protein extraction method, reflected the highest quantified proteins compared to other, traditional methods. SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the whole-body protein reflected definite variations between T. hispida and B. polycresta in fraction number and activity at the two sites. Varied expression levels for metallothionein (MT) heavy metal resistance proteins for B. polycresta and T. hispida were also detected in the study. Based on these results, we suggest that biochemical biomarkers could infer environmental hazards, B. polycresta and T. hispida are successful biomarkers for heavy metal pollution.


Manufacturing ceramic Beetles Esterase isoenzyme Protein fractionation X-ray microanalysis 



The authors are thankful to the Zoology Department, Faculty of Science, Alexandria University.

Authors’ contribution

Both authors designed the study and performed the data collection and analysis. Dalia A. Kheirallah wrote the manuscript. Both authors were involved in the revision of the manuscript.

Compliance with ethical standards

All institutional and national guidelines for the care and use of animals (insects) were followed in conducting this research.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Abu El-Saad, A. M., Kheirallah, D. A., & El-Samad, L. M. (2017). Biochemical and histological biomarkers in the midgut of Apis mellifera from polluted environment at Beheira governorate, Egypt. Environmental Science & Pollution Research, 24(3), 3181–3193.Google Scholar
  2. Aoki, Y. (2011). Metallothionein in insects and their role in tolerance to heavy metals. Japanese Journal of Environmental Toxicology, 14(1), 39–46.Google Scholar
  3. Bochetti, R., & Regoli, F. (2006). Seasonal variability of oxidative biomarkers, lysosomal parameters, metallothioneins and peroxisomal enyzmes in the mediterranean mussel Mytilus galloprovincialis from Adriatic Sea. Chemosphere, 65(6), 913–921.Google Scholar
  4. Brewer, G. J., & Sing, F. G. (1970). An introduction to isozyme techniques. New York: Academic Press.Google Scholar
  5. Butovsky, R. O. (2011). Heavy metals in carabids (Coleoptera, Carabidae). ZooKeys, 100, 215–222.Google Scholar
  6. Callaghan, A. (1989). Genetic and biochemical studies of elevated esterase electro-morphs of Culex pipiens. PhD thesis, University of London.Google Scholar
  7. Cervera, A., Mayomo, A. C., Sendra, M., Martinez-Pardo, R., & Gsrcera, M. D. (2004). Cadmium effects on development and reproduction of Oncopeltus fasciatus (Heteroptera: Lygaeidae). Journal of Insect Physiology, 50(8), 737–749.Google Scholar
  8. Chen, P. (1985). Amino acid and protein metabolism. pp. 177–199. In G. A. Kerkut & L. I. Gilbert (Eds.), Comprehensive insect physiology, biochemistry, & pharmacology (p. 8536). Oxford: Pergamon Press.Google Scholar
  9. Dadd, R. H. (1985). Nutrition: Organisms. (pp. 313–390). In G. A. Kerkut & L. I. Gilbert (Eds.), Comprehensive insect physiology, biochemistry, & pharmacology (p. 8536). Oxford: Pergamon Press.Google Scholar
  10. 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(3), 574–583.Google Scholar
  11. Dutta, T. K., & Kaviraj, A. (1996). Effects of lime acclimation on the susceptibility of two freshwater teleosts and one oligochaete worm to metallic pollutant cadmium. Folia Boilogica (Kraków), 44(3–4), 143–148.Google Scholar
  12. El-Moaty, Z. A., Kheirallah, D. A., & Elgendy, D. A. (2016). Impact of cement dust on some biological parameters of Trachyderma hispida (Coleoptera: Tenebrionidae) inhabiting the vicinity of a cement factory, Mariout region, Alexandria, Egypt. Journal of Entomology & Zoology Studies, 4(5), 797–805.Google Scholar
  13. El-Samad, L. M., Mokhamer, E. H., Osman, W., El-Touhamy, A., & Shonouda, M. (2015). Ecological, histological and biochemical studies on the effect of some environmental pollutants on the herbivorous beetles, Blaps polycresta (Coleoptera: Tenebrionidae). Journal of Advances in Biology, 7(1), 1154–1160.Google Scholar
  14. Gao, H. H., Yan Zhao, H., Yang, J., Zhang, L., Bai, X., & Hu, Z. (2014). Effects of sinc on CarE activities and its gene transcript level in the English grain aphid, Sitobion avenae. Journal of Insect Science, 14(67), 1–11.Google Scholar
  15. Georghiou, G. P., & Pasteur, N. (1978). Electrophoretic esterase patterns in insecticide-resistant and susceptible mosquitoes. Journal of Economic Entomology, 71(2), 201–205.Google Scholar
  16. Ghannem, S., Touaylia, S., & Boumaiza, M. (2017). Beetles (Insecta: Coleoptera) as bioindicators of the assessment of environmental pollution. Human & Ecological Risk Assessment, 24(2), 456–464.Google Scholar
  17. Gintenreiters, S., Ortel, J., & Nopp, H. J. (1993). Effects of different dietary levels of cadmium, lead, copper and zinc on the vitality of the forest pest insect Lymantria dispar L. (Lymantriidae, Lepid). Archives of Environmental Contamination and Toxicology, 25(1), 62–66.Google Scholar
  18. Hensbergen, P. J., Donker, M. H., Van Velzen, M. J., Roelofs, D., Van Der Schors, R. C., Hunziker, P. E., et al. (1999). Primary structure of a cadmium-induced metallothionein from the insect Orchesella cincta (Collembola). European Journal of Biochemistry, 259(1–2), 197–203.Google Scholar
  19. Hoffman, A. A., & Parsons, P. A. (1994). Evolutionary genetics and environmental stress. Oxford. New York: Oxford University Press.Google Scholar
  20. Hogervorst, P. A. M., Wäckers, F. L., & Romeis, J. (2007). Effect of honeydew sugar composition on the longevity of Aphidius ervi. Entomologia Experimentalis et Applicata, 122(3), 223–232.Google Scholar
  21. Holopainen, J., & Oksanen, J. (2018). Arboreal insects as indicators of air pollution effects on Woody plants (pp. 83–96). Publisher: S P B Academic Publ Bv.Google Scholar
  22. Hopkin, S. P. (1989). Ecophysiology of metals in terrestrial invertebrates. London: Elsevier Applied Science.Google Scholar
  23. Hughes, P. R. (1988). Insect populations on host plants subjected to air pollution. In E. A. Heinrichs (Ed.), Plant Stress: Insect Interactions (pp. 249–319).Google Scholar
  24. Hussain, M. S., & Jamil, K. (1992). Appearance of new proteins in water hyacinth weevils (Neochetina eichhorniae), under the influence of metal bioaccumulation. Archives of Environmental Contamination & Toxicology, 22(4), 214–218.Google Scholar
  25. Ilijin, L., Mataruga, P., Radojiei, R., Lazarevil, J., Nenadovil, V., Vlahovil, M., et al. (2009). Effects of cadmium on protocerebral neurosecretory neurons and fitness components in Lymantria dispar L. Folia Boilogica (Kraków), 58(1–2), 9199.Google Scholar
  26. Jemec, A., Drobne, D., Tišler, T., & Sepcic, K. (2010). Biochemical biomarkers in environmental studies-lessons learnt from enzymes catalase, glutathione S-transferase and cholinesterase in two crustacean species. Environmental Science and Pollution Research, 17, 571–581.Google Scholar
  27. Kheirallah, D. A. (2015). Ultrastructure biomarker in Anisops sardeus (Heteroptera: Notonectidae) for the assessment and monitoring of water quality of Al-Mahmoudia Canal, western part of Nile Delta, Egypt. Journal of Bioscience & Applied Research, 1(6), 326–334.Google Scholar
  28. Kheirallah, D. A., Matta, C. A., Yousif, W. B., Sorour, J. M., Shonouda, M. L., Abdel Razik, H. A. (2006). Impact of pollution on the water bug Sphaerodema urinator (Dofour, 1833) inhabiting lakes Mariut and Edku. Ph.D. thesis, Faculty of Science, Alexandria University, Alexandria.Google Scholar
  29. Kheirallah, D. A., El-Moaty, Z. A., & El-Gendy, D. A. (2016). Impact of cement dust on the testis of Trachyderma hispida (Forskal, 1775) (Coleoptra: Tenebrionidae), inhabiting Mariout region (Alexandria, Egypt). Journal of Entomology, 13(3), 55–71.Google Scholar
  30. Khessiba, A., Roméo, M., & Aïssa, P. (2005). Effects of some environmental parameters on catalase activity measured in the mussel (Mytilus galloprovincialis) exposed to lindane. Environmental Pollution, 133(2), 275–281.Google Scholar
  31. Kirkpatrick, L. A., & Feeney, B. C. (2013). A simple guide to IBM SPSS statistics for version 20.0 (Student ed.). Wadsworth: Cengage Learning.Google Scholar
  32. Kozlov, M. V., Wilsey, B. J., Koricheva, J., & Haukioja, E. (1996). Fluctuating asymmetry of birch leaves increases under pollution impact. Journal of Applied Ecology, 33(6), 1489–1495.Google Scholar
  33. Krantzberg, G., & Stokes, P. (1990). Metal concentrations and tissues distribution in larvae of Chironomus with reference to x-ray microprobe analysis. Archives of Environmental Contamination and Toxicology, 19(1), 84–93.Google Scholar
  34. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.Google Scholar
  35. Lagadic, L., Caquet, T., & Ramade, F. (1994). The role of biomarkers in environmental assessment. Invertebrate populations and communities. Ecotoxicology, 3(3), 193–208.Google Scholar
  36. Lambert, O., Veyrand, B., Durand, S., Marchand, P., Bizec, B., Piroux, M., Puyo, S., et al. (2012). Polycyclic aromatic hydrocarbons: Bees, honey and pollen as sentinels for environmental chemical contaminants. Chemosphere, 86(1), 98–104.Google Scholar
  37. Lionetto, M. G., Caricato, R., Giordano, M. E., Pascariello, M. F., 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. Marine Pollution Bulletin, 46(3), 324–330.Google Scholar
  38. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193(1), 265–275.Google Scholar
  39. Magnin, M., Marboutin, E., & Pasteur, N. (1988). Insecticide resistance in Culex quinque fasciatus (Diptera, Culicidae) in West Africa. Journal of Medical Entomology, 25(2), 99–104.Google Scholar
  40. Mazon, L. I., Gonzalez, G., Vicario, A., Estomba, A., & Aguirre, A. (1998). Inhibition of esterases in the marine gastropod Littorina littorea exposed to cadmium. Ecotoxicology and Environmental Safety, 41(3), 284–287.Google Scholar
  41. Monserrat, J. M., Martínez, P. E., Geracitano, L. A., Amado, L. L., Martins, C. M., Pinho, G. L., Chaves, I. S., Ferreira-Cravo, M., Ventura-Lima, J., & Bianchini, A. (2007). Pollution biomarkers in estuarine animals: Critical review and new perspectives. Comparative Biochemistry and Physiology Part C, 146(1–2), 221–234.Google Scholar
  42. Mourente, G., & Díaz-Salvago, E. (1999). Characterization of antioxidant systems, oxidation status and lipids in brain of wild-caught size-class distributed Aristeus antennatus (Risso, 1816) Crustacea, Decapoda. Comparative Biochemistry and Physiology Part B, 124(4), 405–416.Google Scholar
  43. Mukherjeea, S., Mukherjeea, S., Bhattacharyyab, P., & Duttagupta, A. K. (2004). Heavy metal levels and esterase variations between metal-exposed and unexposed duckweed Lemna minor: Field and laboratory studies. Environment International, 30(6), 811–814.Google Scholar
  44. Muller, W. E., Koziol, C., Kurelec, B., Dapper, J., Batel, R., & Rinkevich, B. (1995). Combinatory effects of temperature stress and nonionic organic pollutant on stress protein (HSP 70) gene expression in freshwater sponge, Ephydatia fluviatilis. Environmental Toxicology & Chemistry, 14(7), 1203–1208.Google Scholar
  45. Nation, J. L. (2008). Insect physiology and biochemistry (2nd ed.p. 544). New York: CRC Press.Google Scholar
  46. Niu, C. Y., Jiang, Y., Lei, C. L., & Hu, C. (2002). Effects of cadmium on housefly: Influence on growth and development and metabolism during metamorphosis of housefly. Insect Sci., 9(1), 27–33.Google Scholar
  47. Nunes, B., Carvalho, F., & Guilhermino, L. (2004). Age related chronic effects of clofibrate and clofibric acid on the enzymes acetylcholinesterase, lactate dehydrogenase and catalase of the mosquitofish, Gambusia holbrooki. Chemosphere, 57(11), 1581–1589.Google Scholar
  48. Olson, D., Fadamiro, H., Lundgren, J., & Heimpel, G. E. (2000). Effects of sugar feeding on carbohydrate and lipid metabolism in a parasitoid wasp. Physiological Entomology, 25(1), 17–26.Google Scholar
  49. Osman, W., & Shonouda, M. (2017). X-ray metal assessment and ovarian ultrastructure alterations of the beetle, Blaps polycresta (Coleoptera, Tenebrionidae), inhabiting polluted soil. Environmental Science & Pollution Research, 24(17), 14867–14876.Google Scholar
  50. Osman, W., El-Samad, L. M., Mokhamer, E. H., El-Touhamy, A., & Shonouda, M. (2015). Ecological, morphological, and histological studies on Blaps polycresta (Coleoptera: Tenebrionidae) as biomonitors of cadmium soil pollution. Environmental Science & Pollution Research, 22(18), 14104–14115.Google Scholar
  51. Poirié, M., Raymond, M., & Pasteur, N. (1992). Identification of two distinct amplifications of the esterase B locus in Culex pipiens (L.) mosquitoes from the Mediterranean countries. Biochemical Genetics, 30(1–2), 13–26.Google Scholar
  52. Raymond, M., Qiao, C. L., & Callaghan, A. (1996). Esterase polymorphism in an insecticide susceptible population of the mosquito Culex pipiens. Genetics Research, 67(1), 19–26.Google Scholar
  53. Rebechi, D., Richardi, V., Vicentini, M., Guiloski, I., Silva de Assis, H., & Navarro-Silva, M. (2016). Factors that alter the biochemical biomarkers of environmental contamination in Chironomus sancticaroli (Diptera, Chironomidae). Revista Brasileira de Entomologia, 60, 341–346.Google Scholar
  54. Regoli, F., Niro, M., Chiantore, M., & Winston, G. W. (2002). Seasonal variations of susceptibility to oxidative stress in Adamussium colbeck, a key bioindicator species for the antartic marine environment. Science of the Total Environment, 289(1–3), 205–211.Google Scholar
  55. Saint-Denis, M., Narbonne, J. F., Arnaud, C., & Ribera, D. (2001). Biochemical responses of the earthworm Eisenia fetidaandrei exposed to contaminated artificial soil: Effects of lead acetate. Soil Biology & Biochemistry, 33(3), 1123–1130.Google Scholar
  56. Sainz, A., Grande, J. A., & De la Torre, M. L. (2004). Characterization of heavy metal discharge into the ria of Huelva. Environment International, 30(4), 557–566.Google Scholar
  57. Shonouda, M., & Osman, W. (2018). Ultrastructural alterations in sperm formation of the beetle, Blaps polycresta (Coleoptera: Tenebrionidae) as a biomonitor of heavy metal soil pollution. Environmental Science & Pollution Research, 25(8), 7896–7906.Google Scholar
  58. Sokal, R. R., & Rohlf, F. J. (1981). Biometry: The principles and practice of statistics in biological research (2nd ed.). New York: WH. Freeman.Google Scholar
  59. Stone, D., Jepson, P., & Laskowski, R. (2002). Trends in detoxification enzymes and heavy metal accumulation in ground beetles (Coleoptera: Carabidae) inhabiting a gradient of pollution. Comparative Biochemistry and Physiology Part C, 132(1), 105–112.Google Scholar
  60. Tikul, N., & Srichandr, P. (2010). Assessing the environmental impact of ceramic tile production in Thailand. Journal of the Ceramic Society of Japan, 118(10), 887–894.Google Scholar
  61. Vallejos, C. E. (1983). Enzyme activity staining. In S. D. Tanksley & T. J. Orton (Eds.), Isozymes in plant genetics and breeding, part a (pp. 469–516). Amsterdam: Elsevier.Google Scholar
  62. Van Straalen, N. M., Schobben, J. H., & De Goede, R. G. (1989). Population consequences of cadmium toxicity in soil microarthropods. Ecotoxicology and Environmental Safety, 17(2), 190–204.Google Scholar
  63. Vega-López, A., Galar-Martínez, M., Jiménez-Orozco, F. A., García-Latorre, E., & Domínguez- López, M. L. (2007). Gender related differences in the oxidative stress response to PCB exposure in an endangered goodeid fish (Girardinichthys viviparus). Comparative Biochemistry and Physiology Part A, 46, 672–678.Google Scholar
  64. Vickerman, D. B., & Trumble, J. T. (2003). Biotransfer of selenium: Effects on an insect predator, Podisus maculiventris. Ecotoxicology, 12(6), 497–504.Google Scholar
  65. Welch, W. J. (1993). How cells respond to stress. Scientific American, 268(5), 56–64.Google Scholar
  66. Werner, I., Broeg, K., Cain, D., Wallace, W., Hornberger, M., Hinton, D. E., Koehler, A., et al. (2000). Biomarkers of heavy metal effects in two species of caddisfly larvae from Clark Fork River, Montana: Stress proteins (hsp70) and lysosomal membrane integrity. In: Proceedings of Clark Fork symposium.Google Scholar
  67. Wilczek, G., Babczyńska, A., Migula, P., & Wencelis, B. (2003). Activity of esterases as biomarkers of metal exposure in spiders from the metal pollution gradient. Polish Journal of Environmental Studies, 12(6), 765–771.Google Scholar
  68. Yoshihiko, F. (2004). Life cycle assessment (LCA) study of ceramic products and development of green (reducing the environmental impact) processes. Annual Report of the Ceramics Research Laboratory Nagoya Institute of Technology, Japan, pp. 37–45.Google Scholar

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Authors and Affiliations

  1. 1.Department of Zoology, Faculty of ScienceAlexandria UniversityAlexandriaEgypt

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