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Pollen-inspired enzymatic microparticles to reduce organophosphate toxicity in managed pollinators

Nature Food volume 2, pages 339–347 (2021)Cite this article

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Abstract

Pollinators support the production of the leading food crops worldwide. Organophosphates are a heavily used group of insecticides that pollinators can be exposed to, especially during crop pollination. Exposure to lethal or sublethal doses can impair fitness of wild and managed bees, risking pollination quality and food security. Here we report a low-cost, scalable in vivo detoxification strategy for organophosphate insecticides involving encapsulation of phosphotriesterase (OPT) in pollen-inspired microparticles (PIMs). We developed uniform and consumable PIMs capable of loading OPT at 90% efficiency and protecting OPT from degradation in the pH of a bee gut. Microcolonies of Bombus impatiens fed malathion-contaminated pollen patties demonstrated 100% survival when fed OPT−PIMs but 0% survival with OPT alone, or with plain sucrose within five and four days, respectively. Thus, the detrimental effects of malathion were eliminated when bees consumed OPT−PIMs. This design presents a versatile treatment that can be integrated into supplemental feeds such as pollen patties or dietary syrup for managed pollinators to reduce risk of organophosphate insecticides.

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Fig. 1: A schematic of the passage of microparticles through a bee digestive tract.
Fig. 2: Characterizations of the stability, size distribution and morphology of PIMs.
Fig. 3: Characterizations of OPT encapsulation and activity in PIMs.
Fig. 4: Tracking of digested PIMs by fluorescent imaging of bumblebee GI tracts.
Fig. 5: Characterization of OPT−PIM efficacy through AChE activity assay and bee survival experiments.

Data availability

The data that support the findings of this study are available from the corresponding author on request. Source data are provided with this paper.

References

  1. Abernethy, V. D. Nature’s services: societal dependence on natural ecosystems. Popul. Environ. 20, 277–278 (1999).

    Article  Google Scholar 

  2. Bartomeus, I. et al. Contribution of insect pollinators to crop yield and quality varies with agricultural intensification. PeerJ 2, e328 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Klein, A. M. et al. Importance of pollinators in changing landscapes for world crops. Proc. R. Soc. B 274, 303–313 (2007).

    Article  PubMed  Google Scholar 

  4. Kleijn, D. et al. Delivery of crop pollination services is an insufficient argument for wild pollinator conservation. Nat. Commun. 6, 7414 (2015).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  5. Reilly, J. R. et al. Crop production in the USA is frequently limited by a lack of pollinators. Proc. R. Soc. B 287, 20200922 (2020).

    Article  CAS  PubMed  Google Scholar 

  6. Croft, B. A. Arthropod Biological Control Agents and Pesticides (Wiley, 1990).

  7. Goulson, D., Nicholls, E., Botias, C. & Rotheray, E. L. Bee declines driven by combined stress from parasites, pesticides, and lack of flowers. Science 347, 6229 (2015).

    Article  Google Scholar 

  8. Rissato, S. R., Galhiane, M. S., de Almeida, M. V., Gerenutti, M. & Apon, B. M. Multiresidue determination of pesticides in honey samples by gas chromatography–mass spectrometry and application in environmental contamination. Food Chem. 101, 1719–1726 (2007).

    Article  CAS  Google Scholar 

  9. Ghini, S. et al. Occurrence and distribution of pesticides in the province of Bologna, Italy, using honeybees as bioindicators. Arch. Environ. Contam. Toxicol. 47, 479–488 (2004).

    Article  CAS  PubMed  Google Scholar 

  10. Yao, J., Zhu, Y. C., Adamczyk, J. & Luttrell, R. Influences of acephate and mixtures with other commonly used pesticides on honey bee (Apis mellifera) survival and detoxification enzyme activities. Comp. Biochem. Physiol. C 209, 9–17 (2018).

    CAS  Google Scholar 

  11. Sanchez-Bayo, F. & Goka, K. Pesticide residues and bees—a risk assessment. PLoS ONE 9, e94482 (2014).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  12. Peter, J. V., Sudarsan, T. I. & Moran, J. L. Clinical features of organophosphate poisoning: a review of different classification systems and approaches. Indian J. Crit. Care Med. 18, 735–745 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Fukuto, T. R. Mechanism of action of organophosphorus and carbamate insecticides. Environ. Health Perspect. 87, 245–254 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lerro, C. C. et al. Organophosphate insecticide use and cancer incidence among spouses of pesticide applicators in the agricultural health study. Occup. Environ. Med. 72, 736–744 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Rodriguez, O. P., Muth, G. W., Berkman, C. E., Kim, K. & Thompson, C. M. Inhibition of various cholinesterases with the enantiomers of malaoxon. Bull. Environ. Contam. Toxicol. 58, 171–176 (1997).

    Article  CAS  PubMed  Google Scholar 

  16. Tomlin, C. The Insecticide Manual: A World Compendium (BCPC, 2009).

  17. Pinjari, A. B., Pandey, J. P., Kamireddy, S. & Siddavattam, D. Expression and subcellular localization of organophosphate hydrolase in acephate-degrading Pseudomonas sp strain Ind01 and its use as a potential biocatalyst for elimination of organophosphate insecticides. Lett. Appl. Microbiol. 57, 63–68 (2013).

    Article  CAS  PubMed  Google Scholar 

  18. Zheng, Y., Lan, W., Qiao, C., Mulchandani, A. & Chen, W. Decontamination of vegetables sprayed with organophosphate pesticides by organophosphorus hydrolase and carboxylesterase. Appl. Biochem. Biotechnol. 136, 233–241 (2007).

    Article  CAS  PubMed  Google Scholar 

  19. Benning, M. M., Kuo, J. M., Raushel, F. M. & Holden, H. M. Three-dimensional structure of the binuclear metal center of phosphotriesterase. Biochemistry 34, 7973–7978 (1995).

    Article  CAS  PubMed  Google Scholar 

  20. Roodveldt, C. & Tawfik, D. S. Directed evolution of phosphotriesterase from Pseudomonas diminuta for heterologous expression in Escherichia coli results in stabilization of the metal-free state. Protein Eng. Des. Sel. 18, 51–58 (2005).

    Article  CAS  PubMed  Google Scholar 

  21. McLoughlin, S. Y., Jackson, C., Liu, J. W. & Ollis, D. Increased expression of a bacterial phosphotriesterase in Escherichia coli through directed evolution. Protein Expr. Purif. 41, 433–440 (2005).

    Article  CAS  PubMed  Google Scholar 

  22. Caldwell, S. R., Newcomb, J. R., Schlecht, K. A. & Raushel, F. M. Limits of diffusion in the hydrolysis of substrates by the phosphotriesterase from Pseudomonas diminuta. Biochemistry 30, 7438–7444 (1991).

    Article  CAS  PubMed  Google Scholar 

  23. Hong, S. B. & Raushel, F. M. Metal−substrate interactions facilitate the catalytic activity of the bacterial phosphotriesterase. Biochemistry 35, 10904–10912 (1996).

    Article  CAS  PubMed  Google Scholar 

  24. Naqvi, T. et al. A 5,000-fold increase in the specificity of a bacterial phosphotriesterase for malathion through combinatorial active site mutagenesis. PLoS ONE 9, e94177 (2014).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  25. Benning, M. M., Kuo, J. M., Raushel, F. M. & Holden, H. M. Three-dimensional structure of phosphotriesterase: an enzyme capable of detoxifying organophosphate nerve agents. Biochemistry 33, 15001–15007 (1994).

    Article  CAS  PubMed  Google Scholar 

  26. Singh, B. K. Organophosphorus-degrading bacteria: ecology and industrial applications. Nat. Rev. Microbiol. 7, 156–164 (2009).

    Article  CAS  PubMed  Google Scholar 

  27. Sogorb, M. A., Vilanova, E. & Carrera, V. Future applications of phosphotriesterases in the prophylaxis and treatment of organophosporus insecticide and nerve agent poisonings. Toxicol. Lett. 151, 219–233 (2004).

    Article  CAS  PubMed  Google Scholar 

  28. Yang, C. Y. et al. Improved stability and half-life of fluorinated phosphotriesterase using rosetta. ChemBioChem 15, 1761–1764 (2014).

    Article  CAS  PubMed  Google Scholar 

  29. Rochu, D. et al. Contribution of the active-site metal cation to the catalytic activity and to the conformational stability of phosphotriesterase: temperature- and pH-dependence. Biochem. J. 380, 627–633 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Chen-Goodspeed, M., Sogorb, M. A., Wu, F. Y., Hong, S. B. & Raushel, F. M. Structural determinants of the substrate and stereochemical specificity of phosphotriesterase. Biochemistry 40, 1325–1331 (2001).

    Article  CAS  PubMed  Google Scholar 

  31. Jackson, C. J. et al. Structure-based rational design of a phosphotriesterase. Appl. Environ. Microbiol. 75, 5153–5156 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Pavelka, A., Chovancova, E. & Damborsky, J. Hotspot wizard: a web server for identification of hot spots in protein engineering. Nucleic Acids Res. 37, W376–W383 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Olsen, A. J. et al. Impact of phenylalanines outside the dimer interface on phosphotriesterase stability and function. Mol. Biosyst. 13, 2092–2106 (2017).

    Article  CAS  PubMed  Google Scholar 

  34. Baker, P. J. & Montclare, J. K. Enhanced refoldability and thermoactivity of fluorinated phosphotriesterase. ChemBioChem 12, 1845–1848 (2011).

    Article  CAS  PubMed  Google Scholar 

  35. Malone, L. A., Burgess, E. P. J., Stefanovic, D. & Gatehouse, H. S. Effects of four protease inhibitors on the survival of worker bumblebees, Bombus terrestris L. Apidologie 31, 25–38 (2000).

    Article  CAS  Google Scholar 

  36. Rademacher, E., Harz, M. & Schneider, S. Effects of oxalic acid on Apis mellifera (hymenoptera: apidae). Insects 8, 84 (2017).

    Article  PubMed Central  Google Scholar 

  37. Bailey, L. The action of the proventriculus of the honeybee (Apis mellifera L.). Bee World 32, 92 (1951).

    Article  Google Scholar 

  38. Peng, Y. S. & Marston, J. M. Filtering mechanism of the honey bee proventriculus. Physiol. Entomol. 11, 433–439 (1986).

    Article  Google Scholar 

  39. Roth, R., Schoelkopf, J., Huwyler, J. & Puchkov, M. Functionalized calcium carbonate microparticles for the delivery of proteins. Eur. J. Pharm. Biopharm. 122, 96–103 (2018).

    Article  CAS  PubMed  Google Scholar 

  40. Boyjoo, Y., Pareek, V. K. & Liu, J. Synthesis of micro and nano-sized calcium carbonate particles and their applications. J. Mater. Chem. A 2, 14270–14288 (2014).

    Article  CAS  Google Scholar 

  41. Song, J., Wang, R., Liu, Z. & Zhang, H. S. Preparation and characterization of calcium carbonate microspheres and their potential application as drug carriers. Mol. Med. Rep. 17, 8403–8408 (2018).

    CAS  PubMed  Google Scholar 

  42. Fu, M. F., Wang, A. H., Zhang, X. M., Dai, L. R. & Li, J. B. Direct observation of the distribution of gelatin in calcium carbonate crystals by super-resolution fluorescence microscopy. Angew. Chem. Int. Ed. 55, 908–911 (2016).

    Article  CAS  Google Scholar 

  43. Wszelaka-Rylik, M., Piotrowska, K. & Gierycz, P. Simulation, aggregation and thermal analysis of nanostructured calcite obtained in a controlled multiphase process. J. Therm. Anal. Calorim. 119, 1323–1338 (2015).

    Article  CAS  Google Scholar 

  44. Sukhorukov, G. B. et al. Porous calcium carbonate microparticles as templates for encapsulation of bioactive compounds. J. Mater. Chem. 14, 2073–2081 (2004).

    Article  CAS  Google Scholar 

  45. Griffiths, A. D. & Tawfik, D. S. Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization. EMBO J. 22, 24–35 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Bigley, A. N. & Raushel, F. M. Catalytic mechanisms for phosphotriesterases. Biochim. Biophys. Acta 1834, 443–453 (2013).

    Article  CAS  PubMed  Google Scholar 

  47. Chen, Y. Z., Luo, Z. G. & Lu, X. X. Construction of novel enzyme−graphene oxide catalytic interface with improved enzymatic performance and its assembly mechanism. ACS Appl. Mater. Interfaces 11, 11349–11359 (2019).

    Article  CAS  PubMed  Google Scholar 

  48. Li, P. et al. Nanosizing a metal−organic framework enzyme carrier for accelerating nerve agent hydrolysis. ACS Nano 10, 9174–9182 (2016).

    Article  CAS  PubMed  Google Scholar 

  49. Wang, Y. et al. Rolling circle transcription-amplified hierarchically structured organic−inorganic hybrid RNA flowers for enzyme immobilization. ACS Appl. Mater. Interfaces 11, 22932–22940 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Som, A. et al. Monodispersed calcium carbonate nanoparticles modulate local pH and inhibit tumor growth in vivo. Nanoscale 8, 12639–12647 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  51. Katyal, P., Chu, S. & Montclare, J. K. Enhancing organophosphate hydrolase efficacy via protein engineering and immobilization strategies. Ann. N. Y. Acad. Sci. https://doi.org/10.1111/nyas.14451 (2020).

  52. Arena, M. & Sgolastra, F. A meta-analysis comparing the sensitivity of bees to pesticides. Ecotoxicology 23, 324–334 (2014).

    Article  CAS  PubMed  Google Scholar 

  53. Al Naggar, Y. et al. Organophosphorus insecticides in honey, pollen and bees (Apis mellifera L.) and their potential hazard to bee colonies in Egypt. Ecotoxicol. Environ. Saf. 114, 1–8 (2015).

    Article  CAS  PubMed  Google Scholar 

  54. Martin, M. M. & Lindqvist, L. The pH dependence of fluorescein fluorescence. J. Lumin. 10, 381–390 (1975).

    Article  CAS  Google Scholar 

  55. Cochran, R. in Hayes’ Handbook of Pesticide Toxicology (ed. Krieger, R.) 337−355 (Elsevier, 2010).

  56. Gradish, A. E. et al. Comparison of pesticide exposure in honey bees (hymenoptera: apidae) and bumble bees (hymenoptera: apidae): implications for risk assessments. Environ. Entomol. 48, 12–21 (2019).

    Article  PubMed  Google Scholar 

  57. Peng, Y. S., Nasr, M. E. & Marston, J. M. Release of alfalfa, Medicago sativa, pollen cytoplasm in the gut of the honey bee, Apis mellifera (hymenoptera: apidae). Ann. Entomol. Soc. Am. 79, 804–807 (1986).

    Article  Google Scholar 

  58. Kroon, G., Van Praagh, J. & Velthuis, H. Osmotic shock as a prerequisite to pollen digestion in the alimentary tract of the worker honeybee. J. Apic. Res. 13, 177–181 (1974).

    Article  Google Scholar 

  59. Chaganti, L. K., Venkatakrishnan, N. & Bose, K. An efficient method for FITC labelling of proteins using tandem affinity purification. Biosci. Rep. 38, BSR20181764 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Ellman, G. L., Courtney, K. D., Andres, V. & Featherstone, R. M. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95 (1961).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This material is based on work that are partially supported by the National Institute of Food and Agriculture, US Department of Agriculture, Hatch under 2017-18-107, Counter ACT Program of the National Institute of Health under Award Number R21-NS10383-01 and the National Science Foundation under Award Number IIP-1918981. This work made use of the Cornell Center for Materials Research Shared Facilities which are supported through the NSF MRSEC programme (DMR-1719875).

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Authors and Affiliations

  1. Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY, USA

    Jing Chen, James Webb, Kaavian Shariati & Minglin Ma

  2. Department of Chemical and Biomolecular Engineering, New York University Tandon School of Engineering, Brooklyn, NY, USA

    Shengbo Guo & Jin-Kim Montclare

  3. Department of Entomology, Cornell University, Ithaca, NY, USA

    Scott McArt

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  1. Jing Chen
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Contributions

J.C. and M.M. conceived the study. J.C. and J.W. designed and conducted the experiments. S.G. and J.-K.M. provided the E. coli strain. K.S. drew the schemes. J.C., J.W., J.-K.M., S.M. and M.M. reviewed and interpreted the results. J.C, J.W. and M.M. wrote the manuscript, with input from all authors. All authors reviewed and commented on the manuscript.

Corresponding author

Correspondence to Minglin Ma.

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Competing interests

The technology described in this paper is being licensed to Beemmunity Inc., a start-up company co-founded by J.W. M.M. and J.C. are scientific advisors and shareholders of Beemmunity Inc.

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Peer review information Nature Food thanks Liangfang Zhang, Scott Walper and Scott Medina for their contribution to the peer review of this work.

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Supplementary information

Source data

Source Data Fig. 2

Size distribution, suspension stability and pore size distribution.

Source Data Fig. 3

Protein loading efficiency and relative enzyme activity.

Source Data Fig. 5

Relative activity of AChE and survival data.

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Chen, J., Webb, J., Shariati, K. et al. Pollen-inspired enzymatic microparticles to reduce organophosphate toxicity in managed pollinators. Nat Food 2, 339–347 (2021). https://doi.org/10.1038/s43016-021-00282-0

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