Transepithelial transport of P-glycoprotein substrate by the Malpighian tubules of the desert locust

Autoři: Marta Rossi aff001;  Davide De Battisti aff002;  Jeremy Edward Niven aff001
Působiště autorů: School of Life Sciences, University of Sussex, Falmer, Brighton, United Kingdom aff001;  Department of Bioscience, Swansea University, Swansea, Singleton park, Wales, United Kingdom aff002;  Centre for Computational Neuroscience and Robotics, University of Sussex, Falmer, Brighton, United Kingdom aff003
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0223569


Extrusion of xenobiotics is essential for allowing animals to remove toxic substances present in their diet or generated as a biproduct of their metabolism. By transporting a wide range of potentially noxious substrates, active transporters of the ABC transporter family play an important role in xenobiotic extrusion. One such class of transporters are the multidrug resistance P-glycoprotein transporters. Here, we investigated P-glycoprotein transport in the Malpighian tubules of the desert locust (Schistocerca gregaria), a species whose diet includes plants that contain toxic secondary metabolites. To this end, we studied transporter physiology using a modified Ramsay assay in which ex vivo Malpighian tubules are incubated in different solutions containing the P-glycoprotein substrate dye rhodamine B in combination with different concentrations of the P-glycoprotein inhibitor verapamil. To determine the quantity of the P-glycoprotein substrate extruded we developed a simple and cheap method as an alternative to liquid chromatography–mass spectrometry, radiolabelled alkaloids or confocal microscopy. Our evidence shows that: (i) the Malpighian tubules contain a P-glycoprotein; (ii) tubule surface area is positively correlated with the tubule fluid secretion rate; and (iii) as the fluid secretion rate increases so too does the net extrusion of rhodamine B. We were able to quantify precisely the relationships between the fluid secretion, surface area, and net extrusion. We interpret these results in the context of the life history and foraging ecology of desert locusts. We argue that P-glycoproteins contribute to the removal of xenobiotic substances from the haemolymph, thereby enabling gregarious desert locusts to maintain toxicity through the ingestion of toxic plants without suffering the deleterious effects themselves.

Klíčová slova:

Body fluids – Drosophila melanogaster – Insecticides – P-glycoproteins – Secretion – Locusts – Fluid dynamics – Dye dilution


1. Maddrell SH, Gardiner BO, Pilcher DE, Reynolds SE. Active transport by insect Malpighian tubules of acidic dyes and of acylamides. J Exp Biol. 1974 Oct 1;61(2):357–77. 4443733

2. Ramsay JA. Excretion by the Malpighian tubules of the stick insect, Dixippus morosus (Orthoptera, Phasmidae): amino acids, sugars and urea. J Exp Biol. 1958 Dec 1;35(4):871–91.

3. Phillips JE. Rectal absorption in the desert locust, Schistocerca gregaria Forskal. I. J Exp Biol. 1964 Mar;41:15–38. 14161606

4. O’Donnell M. Insect excretory mechanisms. Adv In Insect Phys. 2008 Jan 1;35:1–122.

5. Williams JC, Hagedorn HH, Beyenbach KW. Dynamic changes in flow rate and composition of urine during the post-bloodmeal diuresis in Aedes aegypti (L.). J Comp Physiol B. 1983 Jun 21;153(2):257–65.

6. Maddrell SH, Klunsuwan S. Fluid secretion by in vitro preparations of the Malpighian tubules of the desert locust Schistocerca gregaria. J Insect Physiol. 1973 Jul 1;19(7):1369–76.

7. Wieczorek HE. The insect V-ATPase, a plasma membrane proton pump energizing secondary active transport: molecular analysis of electrogenic potassium transport in the tobacco hornworm midgut. J Exp Biol. 1992 Nov 1;172(1):335–43.

8. Maddrell SH, Gardiner BO. Excretion of alkaloids by Malpighian tubules of insects. J Exp Biol. 1976 Apr 1;64(2):267–81. 932618

9. Gaertner LS, Murray CL, Morris CE. Transepithelial transport of nicotine and vinblastine in isolated Malpighian tubules of the tobacco hornworm (Manduca sexta) suggests a P-glycoprotein-like mechanism. J Exp Biol. 1998 Sep 15;201(18):2637–45.

10. Karnaky KJ Jr, Petzel D, Sedmerova M, Gross A, Miller DS. Mrp2-like transport of Texas Red by Malpighian tubules of the common American cockroach, Periplaneta americana. Bull Mt Des Isl Biol Lab. 2000;39:52–3.

11. Leader JP, O’Donnell MJ. Transepithelial transport of fluorescent p-glycoprotein and MRP2 substrates by insect Malpighian tubules: confocal microscopic analysis of secreted fluid droplets. J Exp Biol. 2005 Dec 1;208(23):4363–76.

12. O’Donnell MJ, Leader JP. Changes in fluid secretion rate alter net transepithelial transport of MRP2 and P‐glycoprotein substrates in Malpighian tubules of Drosophila melanogaster. Arch Insect Biochem Physiol: Published in Collaboration with the Entomological Society of America. 2006 Nov;63(3):123–34.

13. Wright SH, Dantzler WH. Molecular and cellular physiology of renal organic cation and anion transport. Physiol Rev. 2004 Jul;84(3):987–1049. doi: 10.1152/physrev.00040.2003 15269342

14. Hawthorne DJ, Dively GP. Killing them with kindness? In-hive medications may inhibit xenobiotic efflux transporters and endanger honey bees. PLoS One. 2011 Nov 2;6(11):e26796. doi: 10.1371/journal.pone.0026796 22073195

15. Rheault MR, Plaumann JS, O’Donnell MJ. Tetraethylammonium and nicotine transport by the Malpighian tubules of insects. J Insect Physiol. 2006 May 1;52(5):487–98. doi: 10.1016/j.jinsphys.2006.01.008 16527303

16. Guseman AJ, Miller K, Kunkle G, Dively GP, Pettis JS, Evans JD, Hawthorne DJ. Multi-drug resistance transporters and a mechanism-based strategy for assessing risks of pesticide combinations to honey bees. PLoS One. 2016 Feb 3;11(2):e0148242. doi: 10.1371/journal.pone.0148242 26840460

17. Despland E, Simpson SJ. Food choices of solitarious and gregarious locusts reflect cryptic and aposematic antipredator strategies. Anim Behav. 2005 Feb 1;69(2):471–9.

18. Mainguet AM, Louveaux A, El Sayed G, Rollin P. Ability of a generalist insect, Schistocerca gregaria, to overcome thioglucoside defense in desert plants: tolerance or adaptation?. Entomol Exp Appl. 2000 Mar;94(3):309–17.

19. Sword GA, Simpson SJ, El Hadi OT, Wilps H. Density–dependent aposematism in the desert locust. Proc R Soc Lond B Biol Sci. 2000 Jan 7;267(1438):63–8.

20. Pener MP, Simpson SJ. Locust phase polyphenism: an update. Adv Insect Phys. 2009 Jan 1;36:1–272.

21. Sword GA. Tasty on the outside, but toxic in the middle: grasshopper regurgitation and host plant-mediated toxicity to a vertebrate predator. Oecologia. 2001 Aug 1;128(3):416–21. doi: 10.1007/s004420100666 24549911

22. Sword GA. Density-dependent warning coloration. Nature. 1999 Jan;397(6716):217.

23. Andersson O, Badisco L, Hansen AH, Hansen SH, Hellman K, Nielsen PA, et al. Characterization of a novel brain barrier ex vivo insect‐based P‐glycoprotein screening model. Pharmacol Res Perspect. 2014 Aug;2(4):e00050. doi: 10.1002/prp2.50 25505597

24. Ramsay JA. Active transport of water by the Malpighian tubules of the stick insect, Dixippus morosus (Orthoptera, Phasmidae). J Exp Biol. 1954 Mar 1;31(1):104–13.

25. Eytan GD, Regev R, Oren G, Hurwitz CD, Assaraf YG. Efficiency of P‐glycoprotein–mediated exclusion of rhodamine dyes from multidrug‐resistant cells is determined by their passive transmembrane movement rate. Eur J Biochem. 1997 Aug;248(1):104–12. doi: 10.1111/j.1432-1033.1997.00104.x 9310367

26. Mayer F, Mayer N, Chinn L, Pinsonneault RL, Kroetz D, Bainton RJ. Evolutionary conservation of vertebrate blood–brain barrier chemoprotective mechanisms in Drosophila. J Neurosci. 2009 Mar 18;29(11):3538–50. doi: 10.1523/JNEUROSCI.5564-08.2009 19295159

27. Dermauw W, Van Leeuwen T. The ABC gene family in arthropods: comparative genomics and role in insecticide transport and resistance. Insect Biochemi Mol Biol. 2014 Feb 1;45:89–110.

28. Hamada H, Hagiwara KI, Nakajima T, Tsuruo T. Phosphorylation of the Mr 170,000 to 180,000 glycoprotein specific to multidrug-resistant tumor cells: effects of verapamil, trifluoperazine, and phorbol esters. Cancer Res. 1987 Jun 1;47(11):2860–5. 3567906

29. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nature Methods. 2012 Jun 28;9(7):671. doi: 10.1038/nmeth.2089 22930834

30. R Core Team. R: A language and environment for statistical computing. R J, 2012.

31. Bates D, Mächler M, Bolker B, Walker S. Fitting linear mixed-effects models using lme4. 2014 Jun 23.

32. Akaike H. Information theory and an extension of the maximum likelihood principle. Selected papers of Hirotugu Akaike: Springer; 1998. p. 199–213

33. Kuznetsova A, Brockhoff PB, Christensen RH. lmerTest package: tests in linear mixed effects models. J Stat Softw. 2017;82(13).

34. Forstmeier W, Schielzeth H. Cryptic multiple hypotheses testing in linear models: overestimated effect sizes and the winner’s curse. Behav Ecol Sociobiol. 2011 Jan 1;65(1):47–55. doi: 10.1007/s00265-010-1038-5 21297852

35. Lenth R, Lenth MR. Package ‘lsmeans’. Am Stat. 2018 Nov 2;34(4):216–21.

36. Wickham H. ggplot2: elegant graphics for data analysis. Springer; 2016 Jun 8.

37. Andersson O, Hansen SH, Hellman K, Olsen LR, Andersson G, Badolo L, et al. The grasshopper: a novel model for assessing vertebrate brain uptake. J Pharmacol Exp Ther. 2013 Aug 1;346(2):211–8. doi: 10.1124/jpet.113.205476 23671124

38. Halberg KA, Møbjerg N. First evidence of epithelial transport in tardigrades: a comparative investigation of organic anion transport. J Exp Biol. 2012 Feb 1;215(3):497–507.

39. Tsuruo T, Iida H, Tsukagoshi S, Sakurai Y. Overcoming of vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil. Cancer Res. 1981 May 1;41(5):1967–72. 7214365

40. Al-Qadi S, Schiøtt M, Hansen SH, Nielsen PA, Badolo L. An invertebrate model for CNS drug discovery: Transcriptomic and functional analysis of a mammalian P-glycoprotein ortholog. Biochim Biophys Acta Gen Subj. 2015 Dec 1;1850(12):2439–51.

41. Nielsen PA, Andersson O, Hansen SH, Simonsen KB, Andersson G. Models for predicting blood–brain barrier permeation. Drug Discov Today. 2011 Jun 1;16(11–12):472–5. doi: 10.1016/j.drudis.2011.04.004 21513815

42. Murray CL, Quaglia M, Arnason JT, Morris CE. A putative nicotine pump at the metabolic blood–brain barrier of the tobacco hornworm. J Neurobi. 1994 Jan;25(1):23–34.

43. Ugwu MC, Oli A, Esimone CO, Agu RU. Organic cation rhodamines for screening organic cation transporters in early stages of drug development. J Pharmacol Toxicol Methods. 2016 Nov 1;82:9–19. doi: 10.1016/j.vascn.2016.05.014 27235784

44. Cole SP, Downes HF, Slovak ML. Effect of calcium antagonists on the chemosensitivity of two multidrug-resistant human tumour cell lines which do not overexpress P-glycoprotein. Br J Cancer. 1989 Jan;59(1):42. doi: 10.1038/bjc.1989.9 2569325

45. Eytan GD, Regev R, Oren G, Assaraf YG. The role of passive transbilayer drug movement in multidrug resistance and its modulation. J Biol Chem. 1996 May 31;271(22):12897–902. doi: 10.1074/jbc.271.22.12897 8662680

46. Sharom FJ. The P-glycoprotein efflux pump: how does it transport drugs?. J Membr Biol. 1997 Dec 24;160(3):161–75. doi: 10.1007/s002329900305 9425600

47. Abernethy DR, Schwartz JB. Calcium-antagonist drugs. N Engl J Med. 1999 Nov 4;341(19):1447–57. doi: 10.1056/NEJM199911043411907 10547409

48. MacPherson MR, Pollock VP, Broderick KE, Kean LA, O Connell FC, Dow JA, et al. Model organisms: new insights into ion channel and transporter function L-type calcium channels regulate epithelial fluid transport in Drosophila melanogaster. Am J Physiol. 2001 Feb 1;280(1):C394–407.

49. Paluzzi JP, Yeung C, O’Donnell MJ. Investigations of the signaling cascade involved in diuretic hormone stimulation of Malpighian tubule fluid secretion in Rhodnius prolixus. J Insect Physiol. 2013 Dec 1;59(12):1179–85. doi: 10.1016/j.jinsphys.2013.09.005 24080126

50. Beyenbach KW, Oviedo A, Aneshansley DJ. Malpighian tubules of Aedes aegypti: five tubules, one function. J Insect Physiol. 1993 Aug 1;39(8):639–48.

51. Coast GM. Fluid secretion by single isolated Malpighian tubules of the house cricket, Acheta domesticus, and their response to diuretic hormone. Physiol Entomol. 1988 Dec;13(4):381–91.

52. Bradley TJ. Functional design of microvilli in the Malpighian tubules of the insect Rhodnius prolixus. J Cell Sci. 1983 Mar 1;60(1):117–35.

53. Coast GM, Rayne RC, Hayes TK, Mallet AI, Thompson KS, Bacon JP. A comparison of the effects of two putative diuretic hormones from Locusta migratoria on isolated locust Malpighian tubules. J Exp Biol. 1993 Feb 1;175(1):1–4.

54. Proux JP, Picquot M, Herault JP, Fournier B. Diuretic activity of a newly identified neuropeptide—the arginine-vasopressin-like insect diuretic hormone: use of an improved bioassay. J Insect Physiol. 1988 Jan 1;34(10):919–27.

55. James PJ, Kershaw MJ, Reynolds SE, Charnley AK. Inhibition of desert locust (Schistocerca gregaria) Malpighian tubule fluid secretion by destruxins, cyclic peptide toxins from the insect pathogenic fungus Metarhizium anisopliae. J Insect Physiol. 1993 Sep 1;39(9):797–804.

56. Opitz SE, Müller C. Plant chemistry and insect sequestration. Chemoecology. 2009 Sep 1;19(3):117.

57. Tapadia MG, Lakhotia SC. Expression of mdr49 and mdr65 multidrug resistance genes in larval tissues of Drosophila melanogaster under normal and stress conditions. Cell stress Chaperones. 2005 Mar;10(1):7. 15832942

58. Simões PM, Niven JE, Ott SR. Phenotypic transformation affects associative learning in the desert locust. Curr Biol. 2013 Dec 2;23(23):2407–12. doi: 10.1016/j.cub.2013.10.016 24268415

59. Lanning CL, Fine RL, Corcoran JJ, Ayad HM, Rose RL, Abou-Donia MB. Tobacco budworm P-glycoprotein: biochemical characterization and its involvement in pesticide resistance. Biochim Biophys Acta Gen Subj. 1996 Oct 24;1291(2):155–62.

60. Srinivas R, Udikeri SS, Jayalakshmi SK, Sreeramulu K. Identification of factors responsible for insecticide resistance in Helicoverpa armigera. Comp Biochem Physiol C Toxicol Pharmacol. 2004 Mar 1;137(3):261–9. doi: 10.1016/j.cca.2004.02.002 15171950

61. Akbar SM, Aurade RM, Sharma HC, Sreeramulu K. Mitochondrial P-glycoprotein ATPase contributes to insecticide resistance in the cotton bollworm, Helicoverpa armigera (Noctuidae: Lepidoptera). Cell Biochem Biophys. 2014 Sep 1;70(1):651–60. doi: 10.1007/s12013-014-9969-5 24756730

62. Van Huis A. Strategies to control the Desert Locust Schistocerca gregaria. In: Vreysen MJB, Robinson AS, and Hendrichs J, editors. Area-wide control of insect pests. From research to field implementation. Springer, Dordrecht; 2007. p. 285–296.

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