2 An update on records of Australian ticks

Preface

Attribution Statement

The following chapter has been drafted in accordance with the journal BMC Research Notes.

The following manuscript will be submitted: Egan, S., Evans, M., Fontaine, J., Ryan, U., Irwin, P., and Oskam C. (To be submitted). Australian ticks - Distribution, Hosts and Genetic identification of Ixodida.

The following authors contributed to this manuscript as outlined below³.

Authorship order	Contribution (%)	Concept Development	Data Collection	Data Analyses	Drafting of manuscript
Siobhon L. Egan	80.0	X	X	X	X
Megan Evans	2.5			X
Joseph Fontaine	2.5		X
Una M. Ryan	5.0	X
Peter J. Irwin	5.0	X
Charlotte L. Oskam	5.0	X

By signing this document, the Candidate and Principal Supervisor acknowledge that the information provided is accurate and has been agreed to by all other authors.

__________________       __________________
  Candidate                                 Principal Supervisor

Chapter linking statement:

This chapter is written as a “data resource” to synthesize information about Australian ticks. Curation of records has been performed using data collected from a diverse range of sources. Updated occurrence maps for three highly prevalent species; Amblyomma triguttatum, Ixodes holocyclus and Ixodes tasmani are presented along with an overall map of all 74 tick species present in Australia. This chapter also presents new genetic information for Australian ticks. Molecular “barcodes” are presented for target mitochondrial loci, providing valuable genetic references for species identification. In addition, a high-throughput sequencing assay is also described using pan-Ixodida primers. This provides a high-throughput method to identify ticks by sequencing a ~370 bp product of the mitochondrial 12S rRNA gene on the Illumina MiSeq. This chapter contains information gathered from a range of resources. It is presented in the context of a data centric chapter, and will be submitted to journal BMC Research Notes in the format for a ‘Research Note’.

Discussion of the data presented here is provided however to avoid repetition from the previous chapter (literature review) and in accordance with journal requirements (2000 word limit for main text) the overarching aim here, is to provide a central forum for the synthesis of newly generated and curated data.

Acknowledgment statement: Thank you to Dr. Bruce Halliday (Australian National Insect Collection, CSIRO) for access to records and loan of specimens, Dr. Mark Harvey (Western Australia Museum) and Dr. Owen Seeman (Queensland Museum) for access to records and Prof. Ian Beveridge (Melbourne University) for providing advice on tick records and collections. We acknowledge the use of the Atlas of Living Australia, (https://www.ala.org.au/). We acknowledge that use of records presented in this manuscript has been made possible by the work of previous researchers, we thank them for data collection and curation that has allowed us to conduct this work.

Funding statement: This study was part-funded by the Australian Research Council (LP160100200), Bayer HealthCare (Germany) and Bayer Australia. S.L.E. was supported by an Australian Government Research Training Program (RTP) Scholarship. This project was also part supported by The Holsworth Wildlife Research Endowment & The Ecological Society of Australia (awarded to S.L.E).

Data availability:

The datasets generated during the current study have been made available in the public repositories and the code used in analysis is available on GitHub. Illumina MiSeq data generated from the metabarcoding of ticks targeting the mitochondrial 12S rRNA gene has been deposited in the European nucleotide archive under the project accession number PRJEB46056 (ERP130244), which includes the following sample accession numbers: ERS6635126–ERS6635348 (BioSample # SAMEA8952359–SAMEA8952582). Nucleotide data for a subset of zOTUs generated are available for the molecular identification of ticks and has been uploaded to GenBank under accession numbers MW665133–MW665150. Nucleotide data for multilocus sequence typing (MLST) barcode sequence data produced by Sanger sequencing has been deposited in GenBank under the following accession numbers: OM791407–OM791437 (COI), OM756760–OM756765 (18S rRNA), OM830716–OM830764 (12S rRNA), OM830384–OM830429 (16S rRNA). Code used for analysis and supporting data files used for bioinformatics and statistical analysis are available on GitHub repository github.com/siobhon-egan/wildlife-ticks and the project website.

Ethics: This study was conducted under the compliance of the Australian Code for the Responsibility Conduct of Research (2007) and Australian Code for the Care and Use of Animals for Scientific Purposes, 2013. No specific animal research was conducted for the purposes of this work. Ticks from wildlife were collected as part of established research projects.

Keywords: Ixodida, Amblyomma triguttatum, Ixodes holocyclus, Ixodes tasmani, molecular barcoding

2.1 Abstract

To date 74 tick species have been recorded in Australia which include 60 Ixodidae (hard ticks) and 14 Argasidae (soft ticks). Much of the fundamental knowledge of Australian ticks such as species records and occurrence maps are distributed throughout the scientific literature. This scattered nature can it make it difficult for non-specialists to access basic information, thus, in the present study a synthesis of Australian tick records is provided. A review of tick records and the incidences of humans as hosts, revealed a total of 28 species biting humans. Updated occurrence maps revealed that three widespread tick species Amblyomma triguttatum, Ixodes holocyclus and Ixodes tasmani also represent the most common species identified biting humans. The present study also provides a review of records including curation of those provided in public databases; a companion website also provides interactive maps of these records for readers to investigate. In addition, new genetic data has been generated for mitochondrial loci from Australian ticks. This information has been deposited in a public database (GenBank) and will assist in future efforts that seek to use molecular tools to confirm species identification. The analysis of mitochondrial loci identified the 12S rRNA gene (~370 bp product) was particularly useful at delimitating species. As a result, a high-throughput sequencing assay was developed for high-throughput species identification. The curation of records and genetic data generated here provide new insights in Australian ticks. The findings reported here will be useful to shed new light for future studies on the ecology and systematics of ticks in Australia and be important in assessing public health significance of ticks as vectors for disease.

2.2 Introduction

Ticks (Acari: Ixodida) parasitise a range of vertebrate hosts and serve as vectors for numerous pathogens that affect both humans and animals. Occurrence records of tick species are a valuable tool that is of interest to medical and veterinary industries.

Tick species distribution records provide fundamental information that is required to assess the public health risk in relationship to diseases that ticks can cause. Curation of these records is a pain-staking process and requires extensive searching not only in published journal articles but also in the ‘grey’ literature and unpublished data sets such as those contained within university theses, government reports, etc (Estrada-Pena et al. 2019). Specimen collections housed by museums, universities, and research groups also provide a wealth of information that may not be easily accessible. Online databases, particularly those where members of the public can submit records, can also provide useful information to build occurrence data (Belbin et al. 2021). However, careful consideration of the content of such records may be required where non-experts are providing identifications.

The aim of this study was to provide an update on records of Australian ticks. The present study provides: (i) a list of tick species in Australia and identification of records for human biting ticks; (ii) updated distribution maps for three widespread tick species and an occurrence map with records of all 74 Australian tick species; (iii) a review of host records for the most widespread tick species in Australia, Amblyomma triguttatum; (iv) new genetic data generated from ticks, useful as reference information for identifications; and finally (v) description and validation of a high-throughput assay for molecular identification of species

2.3 Main text

2.3.1 Methods

2.3.1.1 Tick records

Records were sourced from the Australian National Insect Collection (ANIC), the Western Australia Museum (WAM), Cryptick Laboratory tick archive (Murdoch University) and the literature (including a thorough review of grey literature, e.g., government reports). The Atlas of Living Australia was also investigated to identify records of tick species. Due to the known issues with accurate identification of records in the Living Atlas Australia database (Belbin et al. 2021), this data was further investigated. Tick species with occurrence records outside of their known historic distribution area were verified for likelihood of accurate identification.

Host common names and scientific names are included and follow current taxonomic guidelines as described for mammals (S. Jackson and Groves 2015), except where the taxonomic status has since been updated.

Most prevalent species
The three most common species identified from the analysis above were then selected to produce individual species maps. This also included a more detailed curation of records from the grey literature. As the most abundant and widespread species identified, further investigation of sporadic Am. triguttatum was performed to confirm specimen identification. Tick specimens that were not identified to species level in the ANIC Ixodida collection from New South Wales, Victoria and Tasmania were examined. Historically Am. triguttatum records from these regions have been scarce, and a confirmed identification from these areas could vastly expand the recognised distribution of Am. triguttatum. Due to the widespread nature of Am. triguttatum a list of host records was also synthesied for this species.

2.3.1.2 Tick identification

Samples were visualised using an Olympus SZ61 stereomicroscope (Olympus, Centre Valley, PA, United States) with an external Schott KL 1500 LED (Schott AG Mainz, Germany) light source. Photographs of tick specimens were taken using an Olympus SC30 digital camera (Olympus, Centre Valley, PA, United States) and analysis getIT software (Olympus, Centre Valley, PA, United States). Instar, sex, and species was identified using a combination of available morphological keys and species descriptions (F. S. H. Roberts 1970; J. Jackson et al. 2002; Laan, Handasyde, and Beveridge 2011a; Stephen C. Barker and Walker 2014; Kwak et al. 2017). Where possible Am. triguttatum specimens were assigned to subspecies according to that described in F. H. S. Roberts (1962).

2.3.1.3 Genetic analysis of ticks

Sample collection
Specimens in the Cryptick Laboratory tick archive were used for molecular assays and generation of new sequence data. These ticks were collected since 2016 from various sources such as wildlife rehabilitation centres, veterinary clinics and research projects including collection of ticks from the environment or from wildlife. In the case of ticks collected from wildlife, samples were collected as part of the research project described in Chapters 3 and 4.

DNA extraction and sequencing
The complete details of the methods is described in the A.1. In brief, DNA was extracted from ticks and two molecular approaches were used: (i) multi-locus sequence typing (MSLT) assays with Sanger sequencing and (ii) development of a high-throughput assay to identify ticks.

For the MLST assays, primers were used to amplify ticks at the following mitochondrial loci: cytochrome c oxidase subunit I (COX1) (Song et al. 2011), 12S rRNA (Beati and Keirans 2001), 16S rRNA (Lv, Wu, Zhang, Zhang, et al. 2014) (see Table A.1). Purified PCR products were then subjected to Sanger sequencing in the forward and reverse direction.

Following the results from the MLST approach, the ~370 bp product of the 12S rRNA gene was determined as providing optimal results for species delimitation. In addition, the short fragment size made it suitable to transfer the assay onto the Illumina MiSeq platform. In brief, extracted tick DNA was used to build libraries following the 16S Metagenomic Sequencing Library Preparation (Illumina Part # 15044223 Rev. B). The amplicon PCR was carried out using the 12S rRNA gene pan-Ixodida primers, T1B and T2A (Beati and Keirans 2001) (~370 bp product) with Illumina MiSeq adapters (Table A.1). Libraries were then constructed and sequenced on the Illumina MiSeq using v2 chemistry (2 x 250 paired end).

2.3.1.4 Data analysis and bioinformatics

Distribution maps were produced in RStudio (RStudio Team 2015). Maps of Australia were drawn using ozmaps (Sumner 2021), sf (Pebesma 2018) and sp (Bivand, Pebesma, and Gomez-Rubio 2013) R packages.

Sequences obtained from MLST assays were imported into Geneious 10.2.6 (https://www.geneious.com) for quality inspection. Illumina MiSeq data was analysed using a bioinformatic pipeline with the program USEARCH v11 (R. C. Edgar 2010) with zero radius operational taxonomic units generated (zOTUs). All sequences were subject to BLAST analysis (BLASTN 2.11.0+ (Zhang et al. 2000; Morgulis et al. 2008)) against the NCBI nucleotide collection (nt) database to confirm identification.

Full details of analysis methods are available in supplementary material A.1 and at the repository https://github.com/siobhon-egan/wildlife-ticks.

2.3.2 Results and Discussion

2.3.2.1 Australian ticks

A list of all 74 tick species recorded in Australia is presented in Table 2.1 and includes 60 hard ticks and 14 soft ticks. Five tick species are identified as introduced and are associated with domesticated livestock and companion animals; these include two soft ticks Argas persicus (associated with poultry) and Otobius megnini (imported with horses); and three hard ticks, Haemaphysalis longicornis (associated with cattle), Rhipicephalus linnaei (syn Rhipicephalus sanguineus tropical lineage) (associated with dogs) and Rhipicephalus australis (syn Boophilus microplus) (associated with cattle).

Human records
A review of human incidents of tick bite in Australia identified 28 species reported. In many cases records of species biting humans are confined to sporadic single identifications (Table 2.1). It is noted that reporting of a “human” record with ticks reveals little regarding the potential significance for zoonotic tick-borne diseases. The identification of ticks from human hosts does not necessarily distinguish if the tick was attached and actively feeding, or if the tick was simply found crawling on the individual. While this omission might seem trivial it is a key piece of information in establishing an accurate list of host records, particularly where records of the tick-host association are sparse. In addition, it is important to note in regard to tick-borne diseases, the tick vector remains just one of three components in the cycle of tick-borne pathogens; the dynamics of microbe and reservoir host(s) are also requirements for a zoonotic pathogen to become endemic. Identification of incidents, referred to as “tick encounters”, may prove a more useful tool to quantify the risk of tick bites to people. While this field of research has been conducted in the northern hemisphere (Hook et al. 2021), similar studies have not been conducted in Australia.

The list of human-tick records in Australia includes a diverse range of species however, the overwhelming evidence concludes that Ix. holocyclus and Am. triguttatum contribute to the vast majority (>90%) of tick bites (Gofton, Doggett, et al. 2015; Geary et al. 2020). An interesting observation in the literature was the identification of the platypus tick Ixodes ornithorynchi from a human in northern Victoria (Geary et al. 2020). The specimen was submitted from a wildlife carer who had direct contact with platypus. In addition to the 28 species reporting biting human two tick species, Argas lagenoplastis and Ornithodorus macmillani were noted by Geary et al. (2020) from birds’ nests but were not directly associated with biting humans.

This review has provided a synthesis of tick-host records in Australia. It is evident that some publications have reported a “new” host record, however review of extant museum collections or historic data has shown it had been previously recorded. For example, an incidence of Ixodes australiensis biting a human was reported as a first record by Kwak (2018), occurring in October 2017. However, Raby et al. (2016), reported the tick biting a person in May 2006. In addition, we note that Ix. australiensis wwas reported in close association with humans by F. S. H. Roberts (1970), however it was noted the tick was “crawling”. An exhaustive review of all host records is not practical where the data is widely distributed in the literature; this serves to demonstrate the value of curated data, especially where it has the potential to be of public health significance.

Table 2.1: List of tick species recorded from Australia. Human records indicated by colour.

Species	Authority	H (a)	Hosts	Notes
(stump-tailed lizard tick )	Neumann 1907	Y	Reptiles (lizards and snakes)	Similar to
	Neumann 1905	N	Echidna (possibly reptiles)	Inornate species. Very few records, description from long-beaked echidna, (now regionally extinct) in Western Australia (historical). Most similar to .
	Neuman 1899	N	Pigs, rodents	Syn. . Few records of this in Australia.
(Calaby’s goanna tick)	Roberts 1963	N	Reptiles (large monitor lizards)
	Roberts 1953	N	Echidna	Morphologically similar to .
(tropical reptile tick)	Koch 1844	N	Reptiles (lizards and snakes)	Previously (eye-less ). Syn. .
	Keirans, King and Sharrad 1994	N	Reptiles (lizards)
(reptile tick)	Neumann 1899	Y	Reptiles (lizards and snakes)	Similar to ; In addition historical identifications maybe confused with .
(seabird tick)	Neumann 1907	Y	Seabirds	Syn. Roberts 1953.
	Roberts 1953	N	Kangaroos	Very few records, only from “kangaros” in north east QLD.
(snake tick)	Koch 1867	Y	Reptiles (snakes)	Previously thought to be subspecies of . Syn .
	Roberts 1953	N	Echidna
	Hirt 1914	N	Echidna
	Neumann 1899	Y	Wallaby and kangaroos
(ornate kangaroo tick)	Koch 1844	Y	Various mammals (kangaroos)	Four subspecies as described by Roberts 1962.
	Lucas 1878	N	Reptiles (lizards and snakes)	Previously , (eye-less ). Syn .
	Keirans, Bull and Duffield 1996	N	Reptiles (lizards)	Can be confused with and sometimes . is ornamented with a light background coloration, little ornamentation in the scapular areas and ornamentation on all festoons.
(wombat tick)	Schulze 1936	Y	Wombats
(short-beaked echidna tick)	Neumann 1899	N	Echidna	Often confusion with in earlier descriptions. Changes in taxonomy mean many syn. names over time e.g. , , .
	Keirans, King and Sharrad 1994	N	Reptiles (lizards)
(southern reptile tick)	Denny 1843	Y	Reptiles (lizards and snakes)	Female holotype maybe or . Previous indentifications remain elusive as syn. with many species in earlier descriptions (e.g. , , and to name a few). Syn , , .
(central Queensland short-beaked echidna tick)	Roberts 1953 (re-instated by Andrews et al. 2006)	N	Echidna	Although described by Roberts in 1960, later considered a subsp. of and therefore not recognised in Roberts 1970 keys. Andrews et al. 2006 provided more detailed description and raised it to species status.
(goana tick)	Fabricius 1775	Y	Reptiles (lizards ), echidna	Syn. , , .
(wallaby tick)	Nuttall and Warburton 1915	Y	Wallabies and kangaroos	Historical records show it may have been confused with . Syn with , .
(Bremner’s possum tick)	Roberts 1963	N	Possum	Can be confused with or .
(Ooenitz Oriental – Australian bird haemaphysalid)	Warburton and Nuttall 1909	Y	Birds	Presence in Australia is recognised but not well documented.
(bandicoot tick)	Warburton and Nuttall 1909	Y	Various mammals (bandicoots, wallabies, rodents)	Can be confused with or .
	Roberts 1963	N	Wallabies and bandicoot
(bush tick)	Neumann 1901	Y	Various mammals (kangaroos)	Introduced.Syn. Nuttall and Warburton 1915 (record presence in Australia), .
	Hirt 1914	Y	Mammal	Syn with .
(rock-wallaby tick)	Roberts 1970	N	Mammal (wallabies)
(rat tick)	Kohls 1948	N	Mammal (rats and small marsupials)	Can be confused with or .
(Antechinus tick)	Roberts 1960	N	Small mammals (antechinus)
(New Zealand seabird tick)	Neumann 1904	N	Seabirds	Can be confused with .
	Roberts 1960	Y	Mammals (kangaroos, bandicoots, possums)
	Barker 2019	N	Echidna	Distinct horn-like projection on palpal article 1. Some similar to Kohls, 1960 from the long-beaked echidna of Papua New Guinea.
	Roberts 1960	Y	Mammals (possums, bandicoots)	Records of this tick in Australia are sparse (previous confused with ), Roberts 1970 reports just a single specimen, a female from Innisfail north Queensland.
	Neumann 1908	N	Mammals (possums, rodents)
(southern paralysis tck)	Roberts 1960	Y	Various mammals (possums, wallabies, rodents)	Earlier records difficult to conclude due to similarity with .
	Maskell 1885	N	Seabirds
	Warburton and Nuttall 1904	Y	Various mammals (kangaroos, bandicoots, possums, wallabies, rodents)	Similar to .
(mountain pygmy possum tick)	Kwak, Madden and Wicker 2018	N	Mountain pygmy possum
(Hirst’s marsupial tick)	Hassall 1931	Y	Various mammals (possums, wallabies, rodents) and birds	See Laan et al. 2011 for description of immature instars, and further details on adults.
(paralysis tick)	Neumann 1899	Y	Various mammals (kangaroos, bandicoots, possums, wallabies, rodents)
(water rat tick)	Swan 1931	N	Rodents
	Andre and Colas-Belcour 1942	N	Seabirds	Many syns: .
	Arthur 1955	Y	Seabirds
(Kopstein’s bat tick)	Oudemans 1926	N	Bats	Very few records of this ticks in Australia. Roberts 1970 refers to a single Australian records based on personal communication. Record from Tadarida colonicus, Nicholson River Northern Territory.
	Heath and Palma 2017	N	Seabirds	Syn.
	Roberts 1962	Y	Numbat	Syn. pre-1960. See Kwak et al. 2018 for new description of nymph and male and redescription of female.
(platypus tick)	Lucas 1846	Y	Platypus
(bat tick)	Neumann 1906	N	Bats
(common marsupial tick)	Neumann 1899	Y	Various mammals (kangaroos, bandicoots, possums, wallabies, rodents)
(possum tick)	Roberts 1960	N	Mammals (possums, bandicoots)	See McCann et al. 2019. Reported high genetic diversity (2 distinct genotype, may have species status).
	White 1852	N	Seabirds
(numbat tick)	Warburton and Nuttall 1909	N	Numbat
	Nuttall 1916	N	Wombats and macropods	See Weaver 2016 for detailed redescription of this species.
(woylie tick)	Ash et al. 2017	N	Woylie	Most similar to , however these two species can be readily differentiated by dentition and the shape of the scutum, spurs on the coxae, and palpal article 1.
(Australian cattle tick)	Fuller 1899	Y	Cattle	Introduced. Syn. .
(brown dog tick)	Latreille 1806	Y	Dogs	Introduced. Recent taxonomic change in 2021, previously tropical lineage. Since the recently described neotype of the species (See Nava et al 2018), a new name has been proposed based on molecular divergence (see Chandra et al. 2019 and Slapeta et al. 2021).
	Kaiser and Hoogstraal 1974	N	Birds (kestrel)
(Australian fairy martin argasid)	Froggatt 1906	N	Birds (fairy martin)
(Lowryae’s bird argasid)	Kaiser and Hoogstraal 1975	N	Birds (kestrel)
	Hoogstraal and Kaiser 1973	N	Birds (crow)
(fowl tick)	Oken 1818	Y	Domestic poultry	Introduced.
(Robert’s bird tick)	Hoogstraal, Kaiser and Kohls 1968	N	Birds (egrets, domestic poultry)
	Kohls and Hoogstraal 1962	N	Bats (presumed)	Recent change of name (see Mans et al. 2019) previously .
(seabird soft tick)	Neumann 1901	Y	Seabirds	Recent change of name (see Mans et al. 2019) previously .
(Davies’ bat argasid)	Kaiser and Hoogstraal 1973	N	Bats	Recent change of name (see Mans et al. 2019) previously .
(Dewae’s bat argasid)	Kaiser and Hoogstraal 1974	N	Bats	Recent change of name (see Mans et al. 2019) previously .
(ghost bat argasid)	Hoogstraal, Moorhouse, Wolf and Wassef 1977	N	Bats	Recent change of name (see Mans et al. 2019) previously .
(kangaroo soft tick)	Warburton 1926	Y	associated with kangaroos	Despite its common name host records are very sparse, and no records of tick attached to host.
(possum soft tick)	Hoogstraal and Kohls 1966	N	Various (possums, birds)
(spinose ear tick)	Duges 1883	N	Domestic horse	Introduced.
a Reference of human biting ticks sourced from published and grey literature including museum records.

2.3.2.2 Distribution maps

An updated map of curated records for all 74 species of tick present in Australia is presented in Figure 2.1. Records without a species identification or those with missing location data were excluded. A final dataset of 6,282 observation records was used to build the occurrence map (Figure 2.1).

It was noted that two species were not present in any museum record searches. Argas lowryae was described by Kaiser and Hoogstraal (1975), and to the best of the authors’ knowledge it has not been recorded since these initial observations. Another soft tick, the invasive spinose ear tick Otobius mengini, was also not identified from any museum records. Instead, a single observation of the species in Perth, Western Australia, reported by the state government (Mayberry 2003) was used in the occurrence map. To complete the occurrence map, data for missing tick species was sourced from the literature. Where possible, records for species that were outside of their historical distribution were individually inspected.

Atlas of Living Australia data

In comparison to the curated data (Figure 2.1), a map of records based solely on data from Atlas of Living Australia was used to build an occurrence map. This analysis only identified 57 species (50 hard ticks and seven soft ticks). Once records with missing data (i.e., no species level identification) were removed, a total of 2,293 records were identified and used to the build map presented in Figure A.1.

Most prevalent species

The species with the highest number of observations were Am. triguttatum (n = 1,286), Ix. tasmani (n = 1,159), Ix. holocyclus (n = 982), Ix. cornuatus (n = 219), and Bothriocroton auruginans (n = 159). The three most common species were then selected for further analysis, including a more detailed curation of records from the grey literature and individual species maps were produced.

Currently Am. triguttatum is divided into four subspecies. This has remained unchanged since it was established by F. H. S. Roberts (1962). A total of 766 records were identified with subspecies status, of these 738 records had location information. A distribution map of these four subspecies is presented in Figure 2.2. After investigation of “Am. triguttatum” specimens recorded in Tasmania and Victoria, it was determined these were either incorrectly identified or incorrectly entered into databases. In the case of records from Tasmania, these specimens were identified as Bt. hydrosauri, while records from Victoria were assigned instead to either other Amblyomma species or as members of the genus Bothriocroton. An example of incorrect records for this species is evident in the map produced using records collected from Atlas of Living Australia (Figure A.1). Amblyomma triguttatum is a widespread tick present throughout Australia from the south-west coast of Western Australia up to the northeast of Queensland. At present the species is considered absent from Tasmania and Victoria, however an invasive population has established on the Yorke Peninsula in South Australia (McDiarmid et al. 2000).

It is interesting that the distribution map (Figure 2.2) of Am. triguttatum has changed minimally since that drawn by F. H. S. Roberts (1962). It further enforces Roberts’ early hypothesis that these disjunct populations are possibly distinct species. However, the authors note that despite our inspection of many thousands of Am. triguttatum specimens in the present study, no single morphological feature was identified that can exclusively and reliably delimit the sub-species. These findings was outlined in the study by F. H. S. Roberts (1962) and remain unchanged today.

Curation of records for Ix. holocyclus identified the species present along the east coast of Australia (Figure 2.3). It was identified along the coastline of Queensland, New South Wales and Victoria. Ixodes tasmani was identified in all states and territories except the Northern Territory (Figure 2.4). Records of the species in Western Australia were mainly confined to the Southwest corner and most records for this location were sourced from published articles and grey literature as opposed to museum collection records.

Figure 2.1: Occurrence map of all known 74 species of ticks (Acari: Ixodida) present in Australia using record curated data.

Figure 2.2: Occurrence map of the four subspecies recorded in Australia. Only records with a valid subspecies identified are included.

Figure 2.3: Occurrence map of records in Australia.

Figure 2.4: Occurrence map of records in Australia.

2.3.2.3 Host records for Amblyomma triguttatum

A review of host records is available in table 2.2, and identified the majority were from mammalian hosts. Given its wide distribution among the mainland there are still many gaps in ecology and life history of Am. triguttatum. In comparison, similar tick species with a wide distribution in the northern hemisphere (e.g. Ix. ricinus and Ix. scapularis) have been extensively studied (Mihalca and Sándor 2013; Tietjen et al. 2020).

A recognised issue in distribution studies more broadly is the identification of areas that truly represent absence, as opposed to a lack of investigation. Amblyomma triguttatum is considered an exophilic tick and can be classified as a ‘hunter species’. Observations by the authors note that species can be found readily in the environment where there is human activity. In addition, Am. triguttatum is a relatively large tick with unfed adults reaching 3–5 mm in size (F. H. S. Roberts 1962) and is readily identified on hosts. With these factors in mind the authors consider that a lack of Am. triguttatum identified is likely to be a true indicator of its absence, or at least of a small tick population size. We note that in arid areas where flora and fauna studies persist, these have been good sources of records based on museum data obtained in the present study. Therefore, until additional data is made available there is strong support that Am. triguttatum persists in disjunct populations throughout Australia.

Helen P. Waudby et al. (2007) listed the domestic cat as a host and included a reference to F. S. H. Roberts (1970), however after reviewing the suite of Roberts’ tick publications, we have not been able to identify such a record. We note however that a host record from a cat was included in additional data obtained by Helen P. Waudby et al. (2007).

Table 2.2: List of host records for . Abbreviations: Australian National Insect Collection (ANIC); Western Australian Museum (WAM). Where host records were ambiguous (e.g., kangaroo), taxa was assigned to the most commonly recorded species in the locality.

Host common name	Host scientific name	Reference
Class: Mammalia
Monotremata
Platypus	Ornithorhynchus anatinus	Neumann 1901 - see Roberts (1962)
Short-beaked echidna	Tachyglossus aculaetus	Krige et al. 2017; ANIC
Dasyuromorphia
Numbat	Myrmecobius fasciatus	Calaby 1960; ANIC
Peramelemorphia
Greater Bilby	Macrotis lagotis	WAM
Diprotodontia
Koala	Phascolarctos cinereus	Barker & Campelo unpublished - Barker and Walker 2014; Waudby et al. 2007; ANIC
Northern hairy-nosed wombat	Lasiorhinus kreffti	ANIC
Western ringtail possum	Pseudocheirus occidentalis	Clarke 2011
Common brushtail possum	Trichosurus vulpecula	Clarke 2011
Eastern grey kangaroo	Macropus giganteus	Pope et al. 1960; Roberts 1962, 1970; Guglielmone 1990; Cooper et al. 2013; ANIC
Western grey kangaroo	Macropus fuliginosus	Roberts 1962; Waudby et al. 2007; Loh et al. 2018; ANIC; WAM
Red kangaroo	Macropus rufus	Pope et al. 1960; Roberts 1962; Loh et al. 2018; ANIC; WAM
Tammar wallaby	Notamacropus eugenii	Waudby et al. 2007, 2018;ANIC
Bridled nail-tail wallaby	Onychogalea frenata	Turni and Smales 2001; ANIC
Swamp wallaby	Wallabia bicolor	Beveridge et al. 1985; Roberts 1962; ANIC
Agile wallaby	Notamacropus agilis	Roberts 1962; Speare et al. 1983; Cooper et al. 2013; ANIC
Black-striped wallaby	Notamacropus dorsalis	Roberts 1962; ANIC
Red-necked wallaby	Notamacropus rufogriseus	ANIC
Northern Nail-tail wallaby	Onychogalea unguifera	ANIC
Allied Rocky-wallaby	Petrogale assimilis	ANIC
Western brush wallaby	Notamacropus irma	ANIC
Unadorned Rock-wallaby	Petrogale inornata	ANIC
Whiptailed wallaby	Notamacropus parryi	Roberts 1970; ANIC
Spectacled Hare-wallaby	Lagorchestes conspicillatus	ANIC; WAM
Common wallaroo	Osphranter robustus	Roberts 1962; Cooper et al. 2013; ANIC; WAM
Antilopine wallaroo	Osphranter antilopinus	ANIC
Northern bettong	Bettongia tropica	Barker & Campelo unpublished - Barker and Walker 2014
Woylie	Bettongia penicillata	Kaewmongkol et al. 2011; Northover 2019; ANIC
Rufous bettong	Aepyprymnus rufescens	Cooper et al. 2013; ANIC
Primates
Human	Homo sapiens	Roberts 1962; Pearce and Grove 1987; McDiarmid et al. 2000; Andrews et al. 2007; Owen 2007; Gofton et al. 2015; ANIC; WAM
Rodentia
Black rat	Rattus rattus	Waudby et al. 2007
Laboratory / brown rat	Rattus norvegicus	Guglielmone and Moorhouse 1985(a)
Lagomorpha
Rabbit	Oryctolagus cuniculus	Roberts 1953(a); Guglielmone and Moorhouse 1985; Waudby et al. 2007; ANIC(a)
Carnivora
Dog	Canis lupus familiaris	Roberts 1962; Waudby et al. 2007; Andrews et al. 2007; Greay et al. 2016; ANIC; WAM
Cat	Felis catus	Waudby et al. 2007; WAM
Perissodactyla
Horse	Equus caballus	McCarthy 1960; Roberts 1962; Greay et al. 2016; Chalada et al. 2018; ANIC
Artiodactyla
Pig	Sus scrofa	Roberts 1962; Guglielmone 1990; Li et al. 2010; ANIC
Cattle	Bos taurus	Roberts 1962, 1970; ANIC
Swamp buffalo	Babalus bubalis	ANIC
Goat	Capra hircus	Pope et al. 1960
Domestic sheep	Ovis aries	Roberts 1962; ANIC; WAM
Class: Aves
Australian raven	Corvus coronoides	ANIC
Torresian crow	Corvus orru	ANIC
Class: Reptilia
Bobtail/sleepy lizard	Tiliqua rugosa	Waudby et al. 2007; Petney et al. 2008;
a Host record reported from laboratory animals.

2.3.2.4 Molecular barcodes and Systematics

Molecular systematics has become the dominant method of species identification in disciplines including protozoology (Maia, Carranza, and Harris 2016; Maslov et al. 2019) and microbiology (Margos et al. 2019). However, for ectoparasites, including ticks, morphological tools remain the gold standard. Therefore, the splitting of species based solely on molecular information without support of morphological features is unlikely to be useful to the field of tick taxonomy. However, from an evolutionary perspective the molecular information generated here will assist in phylogenetic reconstructions and may assist in refining species boundaries.

New molecular barcodes generated in the present study are presented in Table A.2 which include GenBank accession numbers. An approach to tick taxonomy incorporating both traditional and new technologies is needed and consideration needs to be given to the broader impact of species nomenclature. Particularly, for tick species of medical and veterinary importance, splitting species or a change of name can create confusion and wider uptake can be slow. Compelling evidence for name changes is therefore needed before considering a new taxonomic nomenclature of these species.

The development of molecular information provides a fundamental tool to assist in species identification and systematics. Unlike morphological descriptions and dichotomous keys, molecular barcodes are a characteristic of a species which is shared across all life stages. As studies progress towards adapting an integrative approach in tick taxonomy (F. Dantas-Torres 2018), the use of molecular barcodes, as generated here, will be useful to identify characteristics used to deliminate species.

High-throughput sequencing sequencing

High-throughput sequencing sequencing targeting the 12S rRNA gene was successful at identifying tick species. This included immature life stages where multiple specimens were pooled at the DNA extraction level. Pooling of ticks (especially larvae and nymph stages) is implemented in many molecular studies of tick-borne pathogen or microbial characterisation to increase the throughput of samples (Estrada-Pena et al. 2021). The ability to deliminate species with a short sequence length (~ 370 bp) make this high-throughput assay easily transferable to many short read sequence platforms. The same extracted DNA or RNA used for pathogen detection and be used in the assay presented here to accurately determine species. In particular, it is suited to the identification of immature tick life stages whose identity is ambiguous or where certain morphological features are missing or damaged, which prevent species-level identification. An ability of the 12S rRNA gene to act as a barcoding gene for ticks has been identified in similar studies (Lv, Wu, Zhang, Chen, et al. 2014; Kanduma et al. 2019), and is shown by the level of phylogenetic separation among species shown in Figure 2.5 (see list of accession numbers and species identification in Table A.3). For example morphologically similar species, such as Ix. holocyclus (OM830732) and Ix. cornuatus (OM830728), can be distinguished easily as demonstrated in Figure 2.5 (see also Table A.3).

Figure 2.5: Maximum likelihood (ML) phylogenetic reconstruction of Ixodida ZOTUs based on a 377 bp alignment of the 12S rRNA gene Substitution model K3Pu +F + I + G4 with 10,000 replicates. Node values correspond to bootstrap support where values > 0.7 indicated by shaded circles. Number of substitutions per nucleotide position is represented by the scale bar. Sequences generated in the present study represented in blue (ZOTUs), and red (reference sequences).

2.3.3 Limitations

The nature of using occurrence records to map distribution does have several limitations (Fourcade 2016). However, as ticks are obligate blood feeders which require a host, the nature of their life history makes occurrence data more suitable to map distributions. In particular, where tick species are aggressive human biting species, such as Am. triguttatum (S. R. Graves and Stenos 2017), the absence of records is a useful indicator that the species is likely absent from that area. Alternatively, low levels of human interactions in areas where the tick is present in the environment may also be responsible for absence of records. A comprehensive search strategy was used and attempts were made to use a diverse range of sources (museum collections, public databases, grey literature etc.). Therefore, we accept that it is possible that our occurrence maps may not represent the complete geographical distribution of all 74 tick species present in Australia. Tick species distribution maps included a large portion of data based on historical identifications. While every effort was made to ensure such records were from trusted sources, such as those identified by a suitability qualified person or collections from museum records, it is not possible to verify every single observation. Where records outside of historic distributions or unusually sporadic data points were observed, efforts were made to verify specimen identification.

The generation of new molecular barcodes provided in the present study represents only a portion of the 74 tick species present in Australia. Despite this, the authors feel this information is still valuable to researchers. By making the molecular data public it will be of assistance to future researchers working towards genetic characterisation of ticks and be of use in phylogenetic reconstruction and taxonomic studies.

2.4 Conclusion

While the biology and natural history of ticks is understood in a broad context, much of this is based on data obtained from just a handful of species. The most frequently incriminated human biting ticks in Europe and North America, Ixodes ricinus and Ixodes scapularis, are responsible for the majority of tick-borne infections in those continents (Eisen and Eisen 2018; Gray, Kahl, and Zintl 2021). The life history and ecology of these tick species has been studied and provides important information needed to inform public health measures (Gray, Kahl, and Zintl 2021). In contrast few, if any, tick species in Australia have been studied to the same degree.

With rapid urbanization and the effects of climate change, the interface between humans and ticks is predicted to increase (L. Gilbert 2021). Despite the well documented history of the discovery of Lyme disease in North America during the 1970’s and 1980’s (Ostfeld and Keesing 2000), Australia continues to trail behind the rest of world with respect to knowledge about tick-borne diseases. Without fundamental research into the natural history, ecology and molecular systematics of Australian ticks, the country is ill-equipped to understand the dynamics of potential tick-borne infections.

It is expected that the data presented from this research will provide the necessary foundations to further explore Australian tick systematics. It is important that previous records are met with a healthy dose of criticism and work can begin towards a clearer understanding on Australian ticks.