Berry, Christina M. (2017) Resolution of the taxonomic status of Rhipicephalus (Boophilus) microplus. PhD thesis. http://theses.gla.ac.uk/8165/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten:Theses http://theses.gla.ac.uk/ [email protected] Resolution of the taxonomic status of Rhipicephalus (Boophilus) microplus Christina M. Berry BSc (Hons) PGCE MSc Submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy (PhD) Institute of Biodiversity, Animal Health and Comparative Medicine College of Medical, Veterinary and Life Sciences University of Glasgow 6th January 2017 Abstract Rhipicepahlus (Boophilus) microplus is an obligate feeding, hard tick of great economical importance in the cattle industry. Every year billions of dollars of loss is attributed to R.(B) microplus, mainly through loss of cattle due to pathogens transmitted such as Babesia and Anaplasma, but also through damage to hides from blood-feeding. There is conflicting evidence regarding the taxonomic status of R.(B) microplus, however the most recent published research has been in support of the reinstatement of R.(B) australis as a species distinct from R.(B) microplus. The way in which some members of the scientific community have responded to the designation of separate species has implications for vaccine and acaricide research. In this study, we aimed to resolve the taxonomic status of Rhipicephalus (Boophilus) microplus, using morphological and phylogenetic approaches. 1,650 Rhipicephalus (Boophilus) microplus ticks from Australia, Thailand, South Africa, North and Central America and South America were used in this study. 340 specimens consisting of 170 R.(B) annulatus (USA) and 170 R.(B) decoloratus (South Africa) were also used. To maximize the information obtained from morphological observations, three methods were used; a binary scoring system based on previously described features, a standard morphometric method, and the more novel approach of geometric morphometrics. For the phylogenetic analysis three genes were used; the mitochondrial gene COX1 and two functional nuclear genes; Bm86 and βAOR. Morphological scoring is the process of assigning a binary value to any feature as being present or absent, or satisfying a logical comparator. For this study the scoring matrix was based on previously described sets of morphological criteria used for discriminating among species. Each of the populations for which samples were obtained was tested using four two-way analyses, each of which was designed to test whether a sample should be classified as one of two possible species: R.(B) australis versus R.(B) microplus; R.(B) microplus versus R.(B) annulatus; R.(B) microplus versus R.(B) decoloratus; and R.(B) annulatus versus R.(B) decoloratus. The scoring system was highly repeatable for the differentiation of males and females of R. (B) annulatus and R.(B) decoloratus from both of R.(B) microplus and R.(B) decoloratus. However, in the case of R.(B) australis and R.(B) microplus, clear differentiation was not achieved for either male or female ticks. Among females, the Australian population were classified almost evenly as R.(B) australis and R.(B) microplus, with 8 individuals showing a mixture of features and therefore not able to be classified. Ticks from the rest of the regions were mainly classified as R.(B) microplus, which is to be expected as R.(B) australis is reported in Australia. However, only the Mozo isolates were classified as solely R.(B) microplus. The remaining regions included several ticks with mixed features. Six ticks from South Africa, and four of the Juarez isolate were classified as R.(B) australis. Among the males I an entirely different pattern emerged. Most male ticks from all geographical locations were classified as either R.(B) australis or showed a mixture of both features, with only a small number scoring as R.(B) microplus. Morphometrics is the linear measurement from one anatomical landmark to another and is a widely-used technique for quantifying phenotypic variation. Twelve features based on previous morphometric work were used. The results obtained from this study varied according to stage and sex. For the larvae, the Fisher Pairwise comparison showed that the Australian ticks tended to have a shorter body length, idiosoma length and narrower scutum width. Among the remaining morphological features, there were no consistent patterns in the different populations and species. A principal components analysis (PCA) was undertaken and in PC1 the strongest feature was scutum length and hypostome length. In PC2 the strongest feature was idiosoma length. The PCA of larval stage ticks didn’t provide conclusive evidence that R.(B) australis is a distinct species from R.(B) microplus, and there was no obvious grouping based on region at all, even when including R.(B) decoloratus and R.(B) annulatus. In relation to the male ticks studied, the Fisher Pairwise comparison and the PCA showed that Australian males (presumed R.(B) australis) were significantly different from the other isolates. As with the larvae, no patterns were seen in the other populations, based on species or region. In PC1 palpal length measures were the strongest features for differentiation and in PC2 the length of the ventral basis capituli had the strongest effect. The adult female samples yielded a mixed result. There was no real trend in the size of Australian ticks observed from the Fisher Pairwise comparison. However, R.(B) decoloratus tended to be smaller for most of the morphological features tested and R.(B) annulatus tended to be larger. This observation was inconsistent with the results from the PCA, in which there was grouping of Australian ticks. Measures of palpal length, width of the basis capituli and the length of the dorsal basis capituli were the strongest for differentiation in PC1. In PC2 the length of the ventral basis capituli was the strongest feature for differentiating populations. Geometric morphometrics is the quantitative representation of shape using coordinates in the form of landmarks, instead of measurements and is intended to give the shape of the feature independent of size. Hence it is useful for eliminating the effect of size distortion occurring with physiological changes. Geometric morphometric analysis did not clearly and consistently enable the differentiation of any of the populations of ticks in this study. Each feature differed among samples in different sets of pairwise relationships. Mitochondrial cytochrome oxidase subunit I gene (COX1) has been presented as a suitable mitochondrial gene to clarify complex groupings that were not resolved when using other mitochondrial genes. COX1 has also been proposed to be the main gene for II differentiating between R.(B) microplus and R.(B) australis. The aims of this study were to confirm whether COX1 can be used to resolve complex relationships within the R.(B) microplus clade and to determine whether there is justification for the view that R.(B) australis is a distinct species from R.(B) microplus. Maximum likelihood trees were constructed with a Bootstrap analysis. A relaxed clock Bayesian analysis was then undertaken to estimate topology and divergence timings, using three ticks found in amber covering three genera: Amblyomma, Hyalomma and Ixodes to calibrate the clock. These analyses suggest that R.(B) microplus is a clade, containg five subspecies including R.(B) annulatus, R.(B) australis, and three, regionally based clades of R.(B) microplus: 1. All the South and Central American isolates together with isolates from Cambodia, Thailand, and some of those from Malaysia; 2. Indian and the remaining Malaysian isolates; 3. Most of the Chinese isolates. R.(B) decoloratus shares a common ancestor with R.(B) microplus and R.(B) annulatus however it is clearly divergent, appearing to be more related to R. bursa. All proposed groups of R.(B) microplus also appear to have evolved within the same time scale (within the last 20 million years). Bm86 is the name given to a midgut glycoprotein that is the target antigen of the only commercially available vaccine against ticks. All the prior work on this gene has been conducted using cDNA and suggests a high degree of sequence variation and the presence of different isoforms. The aim was to use genomic DNA to examine the regional variation in the Bm86 sequences and to determine whether Bm86 variation segregated according to the recently proposed taxonomic re-classification of R.(B) microplus and R.(B) australis. After extensive optimization, it was found that all primer sets, including those previously published and those designed in this project, failed on the extracted genomic DNA from all isolates. High variability in the published cDNA sequences indicated an extremely high mutation rate, which could potentially be linked to variation in the function of the protein and its utility as a vaccine immunogen. Analysis of sequence alignments from publicly available databases did not allow grouping of samples by either geographical location or proposed taxon. These findings are in apparent contradiction to claims by other researchers that regional variation in the efficacy of the vaccine is associated with regional variation in sequence. Beta-adrenergic octopamine receptor (βAOR) is a G-protein coupled receptor (GPCR) located on the neuronal cell surface and believed to be the main target of the acaricide amitraz. Polymorphism in βAOR has been associated with amitraz resistance. 121 samples from our isolates were sequenced for a length of 183 base pairs in position 95- 277, among which eight SNPs were identified, five of which had not been previously described.