Population genetics of the cattle tick Rhipicephalus (Boophilus) microplus in Australia

Christian Cutulle (2010). Population genetics of the cattle tick Rhipicephalus (Boophilus) microplus in Australia PhD Thesis, School of Veterinary Science, The University of Queensland.

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Author Christian Cutulle
Thesis Title Population genetics of the cattle tick Rhipicephalus (Boophilus) microplus in Australia
School, Centre or Institute School of Veterinary Science
Institution The University of Queensland
Publication date 2010-03
Thesis type PhD Thesis
Supervisor Dr. Jennifer Seddon
Dr. Nicholas Nils Jonsson
Total pages 157
Total colour pages 6
Total black and white pages 151
Subjects 07 Agricultural and Veterinary Sciences
Abstract/Summary In Australia, the cattle tick Rhipicephalus microplus has become endemic in the tropical and subtropical coastal regions of Western Australia, Northern Territory and Queensland (QLD), with outbreaks occurring in north-eastern areas of New South Wales (NSW). The heavy reliance on the use of acaricides to control this parasite has resulted in the emergence of acaricide resistance to the majority of acaricides, including fluazuron. Acaricide resistance poses a serious threat to the livestock industry and the chemical companies by limiting the effectiveness of chemical tick control measures. Population genetics studies can be performed to understand patterns of tick dispersal and the subsequent spread of resistance, and also to monitor the effectiveness of management strategies that are intended to delay the emergence of resistance. Before this study, there was no information about the population structure of R. microplus in Australia. Therefore, we used thirteen microsatellites to examine the genetic structure of paired susceptible and amitraz-resistant field isolates that were collected from three regions in QLD (southern, northern and central Queensland) and two locations in northern NSW. While all QLD populations exhibited high variability (Ho 0.67-0.74; with 7.00-9.82 alleles per locus), three of the four NSW populations showed very low variability (Ho 0.35-0.48; with 2.36-3.55 alleles per locus). This is unsurprising given that the NSW populations are outbreaks south of a tick quarantine line and so the low variability is likely to be due to strong founder effects and genetic drift. Except for the susceptible population from southern QLD, all sampled populations showed evidence of past bottlenecks, a likely consequence of the use of acaricides. The significant genetic differentiation of most populations indicated limited tick gene flow and hence likely limited cattle exchange, even between locations from the same region across distances as low as 4.2 km. There was no clear pattern of differentiation by region or by resistance status, precluding a conclusion on whether resistance arose independently in each region or was spread with tick dispersal. Parasite mating behaviour has an impact on the parasite’s genetic variation and may affect its population structure. Previous studies showed that R. microplus males could have multiple matings with one or several females; however, multiple paternity was not previously confirmed in this species. Using a highly polymorphic microsatellite marker (BmM1), we were able to confirm multiple paternity among the progeny of ten out of fifteen ticks collected from three cows. The presence of five unique alleles among the progeny, or three unique alleles that could not have been contributed by the female, revealed that the female was successfully fertilised by more than one male. We observed higher heterozygosity levels (average Ho = 0.8; range 0.60 – 1.00) in those progeny sired by more than one male, compared with heterozygosity levels of those sired by a single male (average Ho = 0.73; range 0.28 – 1.00). Multiple paternity reduces the chances of inbreeding and helps to maintain heterozygosity in a population. This observation may contribute to the high diversity we found when we analysed the genetic diversity in two laboratory reference strains (Ho across seven microsatellite loci for NRFS = 0.64; for Ultimo, Ho = 0.65). Although many management strategies have been developed in the past to control this tick, their effectiveness has seldom been tested in the field. The rotation of acaricides is one such strategy and it relies on the principle that a tick resistant to one product is not resistant to the second product and that the effect is augmented where there is a fitness cost associated with resistance to one or other of the products used. Hence, the greatest efficacy of this strategy relies on the existence of a fitness cost. Measurement of fitness under field conditions may be complicated by the confounding effect of gene flow from external susceptible populations. Using seven microsatellites and a SNP that is associated with the expression of synthetic pyrethroid resistance, we genotyped tick larvae that were collected at three specific times (Generation 1, Winter 2004 and Winter 2005) from animals that were initially infested with equal proportions of fully susceptible larvae (NRFS laboratory reference strain) and larvae resistant to amitraz and synthetic pyrethroids (Ultimo laboratory reference strain) and subsequently treated with spinosad, amitraz, and a rotation between these two. Under these different levels of selection, we were able to empirically examine the degree of admixture between the amitraz resistant and susceptible ticks to determine if there was a significant or constant fitness cost associated with amitraz resistance. The frequency of the resistance allele in the populations collected in the first generation (no treatment) ranged from 0.47 to 0.67, however there was not a clear trend in changes to this frequency in later generations, which was expected because the marker was not under selection pressure. A comparison of the level of mortality in the three treatment groups indicated the existence of a fitness cost to amitraz resistance, noted in the rotation groups where the ticks received less than half the number of amitraz treatments than the amitraz groups. The absence of a fitness cost in the populations from the first generation but its presence in populations collected at subsequent time-points suggested a possible relationship between the fitness cost and environmental conditions, possibly related to lower temperatures. Similar levels of admixture related to the relative contribution of the susceptible and resistance strain were found in all the populations and treatments in both Generation 1 (10.0% - 28.6%) and Winter 2004 (36.7% - 50.0%); however, Structure analysis revealed a third cluster in the populations from Winter 2005 that did not follow an specific trend towards the susceptible or resistance strain and likely represented interbreeding between the strains with genetic drift. The molecular confirmation of the fitness cost associated with amitraz resistance, previously detected in the field trials, has implications related to the acaricide control strategies for this tick. Mutations in the para-sodium channel gene in American and Australian R. microplus ticks have been associated with synthetic pyrethroid resistance. In particular, a non-synonymous change at nucleotide position 190, which results in a leucine to isoleucine amino acid substitution (L64I) was previously described in Australian ticks. Using this L64I SNP, we conducted a survey among fifteen field populations from QLD and NSW with different levels of resistance to the synthetic pyrethroids, cypermethrin and flumethrin. There was a strong relationship (adjusted R2 = 95.5%) between homozygous individuals carrying this mutation and their percentage survival after exposure to a discriminating concentration of cypermethrin in a larval packet test. However, the genotyping of these samples revealed that populations that were resistant only to flumethrin did not have the L64I homozygous genotype. Subsequently, we sequenced a 167 bp region that included the domain II S4-5 linker of the same gene in field populations with flumethrin resistance. Importantly, we found a new mutation that resulted in a glycine to valine substitution (G72V). Although the frequency of the G72V homozygous genotype in each flumethrin resistant population was moderately related to its percentage of survival to flumethrin in the larval packet test (adjusted R2 = 73.5%), this relationship was stronger when heterozygous individuals carrying both L64I and G72V were added to G72V homozygotes individuals (adjusted R2 = 93.4%). This stronger relationship suggested an interaction between the two mutations in the same gene such that flumethrin resistance might be conferred by either two copies of the G72V mutation or by being a L64I and G72V heterozygote.
Keyword Rhipicephalus (Boophilus) microplus, cattle tick, population genetics, acaricide resistance, amitraz, synthetic pyrethroid
Additional Notes Pages to be printed in colour: 13, 19 , 23, 55, 62, 142 Pages to be printed in landscape: 16, 33 , 60, 75, 86, 90, 104, 107, 136, 137, 138, 139, 140

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Created: Thu, 16 Dec 2010, 06:15:44 EST by Mr Christian Cutulle on behalf of Library - Information Access Service