Abstract
The embryonation of nematode eggs has been shown to increase their resistance to anthelmintics when parasites are submitted to egg hatch assays. Nevertheless, no mechanism has been suggested to explain this phenomenon. Earlier observations by other authors showed that the biochemical composition of eggshells is altered during the embryonation of eggs. The functional consequences of these changes have not been identified. We studied the changes in membrane environment (eggshells) of Haemonchus contortus eggs during the embryonation by fluidity measurements and their effects on nonspecific mechanisms of resistance to anthelmintics. We previously demonstrated that these mechanisms imply P-glycoproteins (Pgp) belonging to the multi-drug resistance (MDR) system and that the Pgp activity is very susceptible to their lipidic environment. The results obtained here show that the embryonation induced a significant and gradual increase in eggshell fluidity which was associated with an increased resistance to anthelmintics. Differences were observed between H. contortus isolates with various levels of resistance which might result from their specific biology and/or membrane composition. The membrane environment changes could act both on the solubilization of anthelmintics into the eggs and on the efflux of these lipophilic molecules by Pgp.
Similar content being viewed by others
References
Al-Shawi MK, Polar MK, Omote H, Figler RA (2003) Transition state analysis of the coupling of drug transport to ATP hydrolysis by P-glycoprotein. J Biol Chem 278:52629–52640
Beaumont-Schwartz C, Kerboeuf D, Hubert J (1987) Méthodes de mise en évidence de souche de strongles gastro-intestinaux resistantes aux anthelminthiques. Rec Med Vet 163:683–688
Beugnet F, Gauthey M, Kerboeuf D (1997) Partial in vitro reversal of benzimidazole resistance by the free-living stages of Haemonchus contortus with verapamil. Vet Rec 141:575–576
Coles GC (1990) Recent advances in laboratory models for evaluation of helminth chemotherapy. Br Vet J 146:113–119
Driscoll M, Dean E, Reilly E, Bergholz E, Chalfie M (1989) Genetic and molecular analysis of a Caenorhabditis elegans beta-tubulin that conveys benzimidazole sensitivity. J Cell Biol 109:2993–3003
Garrigues A, Escargueil AE, Orlowski S (2002) The multidrug transporter, P-glycoprotein, actively mediates cholesterol redistribution in the cell membrane. Proc Natl Acad Sci U S A 99:10347–10352
Giraud MN, Motta C, Boucher D, Grizard G (2000) Membrane fluidity predicts the outcome of cryopreservation of human spermatozoa. Hum Reprod 15:2160–2164
Gottesman MM, Fojo T, Bates SE (2002) Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer 2:48–58
Jaskoski BJ, Colucci AV (1964) In vitro hatching of Ascaris suums eggs. Trans Am Microsc Soc 83:294–300
Kerboeuf D, Aycardi J (1999) Unexpected increased thiabendazole tolerance in Haemonchus contortus resistant to anthelmintics by modulation of glutathione activity. Parasitol Res 85:713–718
Kerboeuf D, Chambrier P, Le Vern Y, Aycardi J (1999) Flow cytometry analysis of drug transport mechanisms in Haemonchus contortus susceptible or resistant to anthelmintics. Parasitol Res 85:118–123
Kerboeuf D, Guegnard F, Le Vern Y (2002) Analysis and partial reversal of multidrug resistance to anthelmintics due to P-glycoprotein in Haemonchus contortus eggs using Lens culinaris lectin. Parasitol Res 88:816–821
Kerboeuf D, Guegnard F, Vern YL (2003) Detection of P-glycoprotein-mediated multidrug resistance against anthelmintics in Haemonchus contortus using anti-human mdr1 monoclonal antibodies. Parasitol Res 91:79–85
Klein C, Pillot T, Chambaz J, Drouet B (2003) Determination of plasma membrane fluidity with a fluorescent analogue of sphingomyelin by FRAP measurement using a standard confocal microscope. Brain Res Brain Res Protoc 11:46–51
Kremer JJ, Sklansky DJ, Murphy RM (2001) Profile of changes in lipid bilayer structure caused by beta-amyloid peptide. Biochemistry 40:8563–8571
Kwa MS, Kooyman FN, Boersema JH, Roos MH (1993) Effect of selection for benzimidazole resistance in Haemonchus contortus on beta-tubulin isotype 1 and isotype 2 genes. Biochem Biophys Res Commun 191:413–419
Kwa MS, Okoli MN, Schulz-Key H, Okongkwo PO, Roos MH (1998) Use of P-glycoprotein gene probes to investigate anthelmintic resistance in Haemonchus contortus and comparison with Onchocerca volvulus. Int J Parasitol 28:1235–1240
Lagerquist C, Beigi F, Karlen A, Lennernas H, Lundahl P (2001) Effects of cholesterol and model transmembrane proteins on drug partitioning into lipid bilayers as analysed by immobilized-liposome chromatography. J Pharm Pharmacol 53:1477–1487
Lincke CR, Broeks A, The I, Plasterk RH, Borst P (1993) The expression of two P-glycoprotein (pgp) genes in transgenic Caenorhabditis elegans is confined to intestinal cells. EMBO J 12:1615–1620
Luker GD, Flagg TP, Sha Q, Luker KE, Pica CM, Nichols CG, Piwnica-Worms D (2001) MDR1 P-glycoprotein reduces influx of substrates without affecting membrane potential. J Biol Chem 276:49053–49060
Mansfield LS, Gamble HR, Fetterer RH (1992) Characterization of the eggshell of Haemonchus contortus. I. Structural components. Comp Biochem Physiol B Biochem Mol Biol 103:681-686
Mukhopadhyay K, Kohli A, Prasad R (2002) Drug susceptibilities of yeast cells are affected by membrane lipid composition. Antimicrob Agents Chemother 46:3695–3705
Pallares-Trujillo J, Lopez-Soriano FJ, Argiles JM (2000) Lipids: a key role in multidrug resistance?. Int J Oncol 16:783–798
Qu Q, Chu JW, Sharom FJ (2003) Transition state P-glycoprotein binds drugs and modulators with unchanged affinity, suggesting a concerted transport mechanism. Biochemistry (Mosc) 42:1345–1353
Riou M, Guegnard F, Le Vern Y, Kerboeuf D (2003) Modulation of the multidrug resistance (MDR) system in the nematode Haemonchus contortus by changing cholesterol content: effects on resistance to anthelmintics. J Antimicrob Chemother 52:180–187
Rogers WP (1958) The physiology of the hatching of eggs of Ascaris lumbricoides. Nature 181:1410–1411
Rogers WP (1960) The physiology of infective processes of nematodes parasites: the stimulus from the animal host. Proc R Soc B 152:367–386
Rogers WP (1966) Exhshealment and hatching mechanisms in helminths. In: Soulsby EJL (ed) Biology of parasites. Emphasis on veterinary parasites. Academic Press, Philadelphia, pp 33–40
Rothnie A, Theron D, Soceneantu L, Martin C, Traikia M, Berridge G, Higgins CF, Devaux PF, Callaghan R (2001) The importance of cholesterol in maintenance of P-glycoprotein activity and its membrane perturbing influence. Eur Biophys J 30:430–442
Sangster NC (1994) P-glycoproteins in nematodes. Parasitol Today 10:319–322
Sharom FJ (1997) The P-glycoprotein efflux pump: how does it transport drugs? J Membr Biol 160:161–175
Shechter E, Rossignol B (1997) Biochimie et biophysique des membranes. Aspects structuraux et fonctionnels, 2nd edn. Dunod, Paris
Snedecor GW, Cochran WG (1982) Statistical methods, 7th edn. Iowa State University Press, Ames
Wang EJ, Casciano CN, Clement RP, Johnson WW (2000) Two transport binding sites of P-glycoprotein are unequal yet contingent: initial rate kinetic analysis by ATP hydrolysis demonstrates intersite dependence. Biochim Biophys Acta 1481:63–74
Acknowledgements
The authors thank Dr. Yan Van Wyk, Dr. Gilles Aumont and Professor Roger Prichard for kindly providing the H. contortus isolates, Limouzin and his team for the careful maintenance of animals and Marie-Colette Fauré and her team for their generous help with the bibliography. We also thank the “Region Centre” for financing this research. The experiments comply with the current French laws on animal experimentation.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Riou, M., Koch, C. & Kerboeuf, D. Increased resistance to anthelmintics of Haemonchus contortus eggs associated with changes in membrane fluidity of eggshells during embryonation. Parasitol Res 95, 266–272 (2005). https://doi.org/10.1007/s00436-004-1288-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00436-004-1288-0