RESISTANCE TO EMAMECTIN BENZOATE IN SEA LICE A Thesis Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Department of Biomedical Sciences Atlantic Veterinary College University of Prince Edward Island Okechukwu Obiora Igboeli Charlottetown, P.E.I. March, 2013 © 2013. O. O. Igboeli i CONDITION OF USE The author has agreed that the Library, University of Prince Edward Island, may make this thesis freely available for inspection. Moreover, the author has agreed that permission for extensive copying of this thesis for scholarly purposes may be granted by the professor or professors who supervised the thesis work recorded herein or, in their absence, by the Chairman of the Department or the Dean of the Faculty in which the thesis work was done. It is understood that due recognition will be given to the author of this thesis and to the University of Prince Edward Island in any use of the material in this thesis. Copying or publication or any other use of the thesis for financial gain without approval by the University of Prince Edward Island and the author’s written permission is prohibited. Requests for permission to copy or to make any other use of material in this thesis in whole or in part should be addressed to: Chair of the Department of Biomedical Sciences Faculty of Veterinary Medicine University of Prince Edward Island Charlottetown, P. E. I. Canada C1A 4P3 ii PERMISSION TO USE POSTGRADUATE THESES Title of thesis: RESISTANCE TO EMAMECTIN BENZOATE IN SEA LICE Name of Author: Okechukwu Obiora Igboeli Department: Biomedical Sciences Degree: Doctor of Philosophy Year: 2013 In presenting this thesis in partial fulfillment of the requirements for a postgraduate degree from the University of Prince Edward Island, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the professor or professors who supervised my thesis work, or, in their absence, by the Chair of the Department or the Dean of the Faculty in which my thesis work was done. It is understood any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Prince Edward Island in any scholarly use which may be made of any material in my thesis. Signature: Address: Dept. of Biomedical Sciences Atlantic Veterianry College University of Prince Edward Island 550 University Avenue Charlottetown, PE C1A 4P3 CANADA Date: March 25, 2013 iii University of Prince Edward Island Faculty of Veterinary Medicine Charlottetown CERTIFICATION OF THESIS WORK We, the undersigned, certify that Okechukwu Obiora Igboeli candidate for the degree of Doctor of Philosophy has presented her/his thesis with the following title: RESISTANCE TO EMAMECTIN BENZOATE IN SEA LICE that the thesis is acceptable in form and content, and that a satisfactory knowledge of the field covered by the thesis was demonstrated by the candidate through an oral examination held on Examiners Dr. Luis Bate Dr. John Burka Dr. Mark Fast Dr. Shona Whyte Dr. Spencer Greenwood Dr. Roger Prichard Date iv ABSTRACT Effective control of parasitic sea lice (including Lepeophtheirus salmonis and Caligus elongatus), is a major challenge currently facing sea cage salmon aquaculture. Emamectin benzoate (EMB; SLICE®), a macrocyclic lactone (ML), has been the drug of choice for sea lice treatment over the past decade due to its ease of administration, as well as efficacy, on all parasitic stages of the salmon parasite. This over-reliance led to increased tolerance to the drug and a consequent decline in its use. Macrocyclic lactone resistance has been linked to ATP- binding cassette (ABC) transporters such as P-glycoprotein (P-gp) in arthropods and nematodes. The present study investigated the role of P-gp in sea lice resistance to EMB. Analysis of bioassay results indicated a 4 to 26 fold higher EMB EC50 for L. salmonis samples collected from salmon farms in the Bay of Fundy, New Brunswick (NB), in 2011 compared with a similar study carried out between 2002 and 2004, suggesting loss of EMB efficacy in sea lice in this region. An assay for ATPase activity (utilizing SB-MDR1-Sf9 membranes over-expressing P-gp), as well as a competitive inhibition test, showed that EMB interacts with this transporter. Emamectin benzoate caused a significant concentration-dependent increase in P-gp mRNA expression in L. salmonis. Also, results indicated a temporal increase in P-gp mRNA levels in archived L. salmonis collected from 2002 to 2011. These findings suggest that EMB is a substrate for P-gp, will induce higher expression of the transporter, and that the latter is likely involved in loss of EMB efficacy in L. salmonis. Contrary to initial prediction that prior host immunostimulation would enhance EMB efficacy in the parasite, sea lice attached to salmon with a history of immunostimulation exhibited greater survival and significantly higher P-gp mRNA expression compared with control groups (P < 0.05). Prior host immunostimulation v likely caused increased expression of P-gp which could have consequently caused decreased EMB efficacy. The present study also investigated (using bioassays and RT-qPCR) reports of differences in sea lice EMB sensitivity in salmon farming regions in the Bay of Fundy. Lepeophtheirus salmonis collected from Grand Manan [Bay Management Area (BMA) 2b] recorded >2 fold lower EMB EC50 values compared with BMAs 1 and 2a populations, confirming the presence of relatively EMB-sensitive sea lice in BMA 2b. Laboratory-reared sea lice maintained their EMB sensitivity status for up to three filial generations. Caligus elongatus, collected from BMA 2a, also showed >2 fold lower EMB EC50 values compared with L. salmonis collected from the same site, indicating species differences in sensitivity to the parasiticide. Attempts to localize sea lice P-gp protein and mRNA in situ using immunohistochemistry and in situ hybridization techniques, respectively, were not successful. Similarly, the quantification of P-gp in L. salmonis using ELISA and Western blot techniques was unsuccessful. This could be due to low P-gp expression at mRNA and protein levels and/or poor antigenic specificity of the antibodies used. Results in this study strongly support the hypothesis that the efflux transporter, P-gp, is involved in reduced sensitivity of L. salmonis to EMB, and that this occurs over time due to drug selection of resistant strains of the parasite. However, there is a need to develop sensitive tools, e.g. probes and antibodies, which will localize and quantify L. salmonis P-gp mRNA and protein production. This will allow for a greater understanding of the role of P-gp in sea lice resistance to EMB as well as a greater ability to monitor resistance development to the parsiticide. Changes in the expression of resistance-associated genes, such as those for P-gp, can be monitored and used in the diagnosis of resistance development to parasiticides. Knowledge of vi the timing for resistance development will inform necessary changes to treatment options to prevent treatment failure. Further comparisons between EMB-sensitive and -resistant strains of L. salmonis may be necessary to verify the degree to which P-gp is involved in the loss of parasite sensitivity to the drug. More investigations on the nature of the species differences in EMB sensitivity in sea lice, as well as the heredity of EMB resistance mechanisms, is necessary for a better understanding of how this parasite strives to survive its control. vii ACKNOWLEDGEMENTS I am eternally grateful to God for granting me the opportunity to achieve this great milestone, sustaining me all the way to the end. To Him be all the praise and glory for ever and ever. Amen. My profound appreciation goes to my supervisors, Drs John Burka and Mark Fast, for their unflinching support and assistance to me all through the program and teaching me to think ‘outside the box’. I wish to thank members of my supervisory committee, Drs Glenda Wright, Collins Kamunde, Jillian Westcott and Ahmed Siah for their invaluable contribution towards the success of this work. I am particularly grateful to Dr. Collins Kamunde for his ardent support to me all through the program. To my mum, Felicia, brothers, Chukwuemeka, Chukwudi, Chinedu and Chukwunonso, and sisters, Adeze Ojukwu and Obianuju Anonye, thank you so much for all your love and support; I could not have faced the challenges I encountered without your prayers and encouragement. To the love of my life, Hope George, thank you so much for your care and support; could not have made it without you, and I love you so much. I am indebted to my Island family- Lynn Levy, Daniel Ugwuja, John Onukwufor, Peter & Dianne Fenton, Priyanka Pundir, Soraya Sayi, Prof Basil & Mrs Joy Ikede, Jeff & Laura Cofran, Daniel & Sunny Hartwig, and St. Paul’s Church Charlottetown for making my stay on the Island a very warm and memorable experience. I am very grateful to Novartis Animal Health and Innovation PEI for granting me Ph.D. Fellowship, and to NSERC for funding the research. Huge thanks to Dr. Harry Murray as well as Monique Saleh, Kathryn Dau-Schmidt, Dr. Sara Purcell and Joy Knight for their technical assistance in the laboratory. viii Table of Contents Title Page ..................................................................................................................................