# Reevaluating the presence of Rhipicephalus australis (Acari: Ixodidae) in Southeast Asia: A phylogenetic approach based on Cambodian tick samples

Didot Budi Prasetyo, Sony Yean, Sébastien Boyer

► To cite this version:

Didot Budi Prasetyo, Sony Yean, Sébastien Boyer. Reevaluating the presence of Rhipicephalus australis (Acari: Ixodidae) in Southeast Asia: A phylogenetic approach based on Cambodian tick samples. Ticks and Tick-borne Diseases, 2025, 16 (3), pp.102478. 10.1016/j.ttbdis.2025.102478. hal-05070269

HAL Id: hal-05070269

https://hal.science/hal-05070269v1

Submitted on 16 May 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

Short communication

# Reevaluating the presence of *Rhipicephalus australis* (Acari: Ixodidae) in Southeast Asia: A phylogenetic approach based on Cambodian tick samples

Didot Budi Prasetyo<fnref n="1" />,*, Sony Yean<fnref n="1" />, Sébastien Boyer

Medical and Veterinary Entomology Unit, Institut Pasteur du Cambodge, No 5 Monivong Blvd., P.O Box 983, Phnom Penh, 12201, Cambodia

## ARTICLE INFO

### Keywords:

Ticks

Rhipicephalus microplus complex

Rhipicephalus australis

Cox1 DNA barcoding

Southeast Asia

Cambodia

## ABSTRACT

Morphological variability between *Rhipicephalus australis* and *R. microplus* has led to taxonomic ambiguity, leading to species misidentification. *Rhipicephalus australis* is reported to have a distribution range in Pacific Ocean region extending to several Southeast Asian countries, including Cambodia, although its presence in continental Southeast Asia has not been supported by molecular data. With growing evidence of conflicting morphological characters, this study aimed to evaluate the presence of *R. australis* in Cambodia using both morphological and molecular identification. Tick specimens were collected from cattle across 21 provinces of Cambodia, and a subset of 95 *R. microplus* complex (37 morphologically identified as *R. australis*, 39 *R. microplus*, and 19 nymphs) was selected for molecular analysis. DNA barcoding of the cox1 gene was performed, and a maximum likelihood phylogenetic tree revealed that all specimens clustered within *R. microplus* clade A. These findings, along with previous observations from other regions, suggest that, in the absence of molecular data, there is no definitive evidence to support the presence of *R. australis* in continental Southeast Asia, particularly in Cambodia.

## 1. Introduction

The *Rhipicephalus microplus* complex is a group of tick species that commonly infest bovids (Barker and Walker, 2014; Walker et al., 2014). They are of veterinary and public health importance due to their ability to transmit pathogens to both animals, mostly livestock, and humans (Tan et al., 2021; Ybañez et al., 2013). This species complex is challenging to distinguish morphologically, but analyses of mitochondrial genes as markers determined five phylogenetically distinct clades: *R. australis*, *R. annulatus*, and three *R. microplus* clades (clade A, B, and C) (Burger et al., 2014; Low et al., 2015). The *Rhipicephalus microplus* complex is widely distributed worldwide (Burger et al., 2014). However, a more detailed examination of the five clades provides greater insight into their regional distribution. *Rhipicephalus microplus* clade A has been recorded in various regions, including South America, Southeast Asia, Africa, and North America (Burger et al., 2014; Roy et al., 2018). Clade B has been predominantly identified in China and in India (Burger et al., 2014), while clade C has been documented in South and Southeast Asian countries, such as India, Thailand, Malaysia, Myanmar, Pakistan, and Bangladesh (Low et al., 2015; Roy et al., 2018; Tanrawatpan et al.,

2022). On the other hand, *R. australis* has been described in Australia, New Caledonia, Indonesia, Philippines, New Guinea, Tahiti and Cambodia (Estrada-Peña et al., 2012). Finally, *R. annulatus* has been recorded on various continents, including Southern Europe, West and Central Asia, North and Sub-saharan Africa, and the Americas (Texas-Mexico border) (Burger et al., 2014). In Southeast Asia, *R. annulatus* has only been recorded in Vietnam (Chien et al., 2016; Hornok et al., 2024).

The species status of *R. australis* was reinstated in 2012 by Estrada Peña et al., based on molecular evidence from specimens collected in Australia, New Caledonia and Indonesia (Estrada-Peña et al., 2012). They also provided a set of morphological characters to distinguish them from *R. microplus*. While the genetic data showed clear separation of the two species, the morphological characters to distinguish them are inconsistent and problematic. A study in Malaysia reported that specimens morphologically identified as *R. australis* were actually assigned to *R. microplus* clade A through cox1 DNA barcoding (Kazim et al., 2022). This discrepancy between morphological and molecular identification has also been reported in other countries, where *R. microplus* clade A and clade C specimens possess characteristics similar to *R. australis* (Low

<footnote>* Corresponding author.
E-mail address: dbprasetyo@pasteur-kh.org (D.B. Prasetyo).</footnote>

<footnote n="1">¹ These authors contributed equally to this work.</footnote>
<figure><img src="image_3.png" /><figcaption>Fig. 1. Distribution of *Rhipicephalus microplus* complex ticks collected in this study based on morphological identification.</figcaption></figure>

et al., 2015; Roy et al., 2018). In the wake of these growing disagreements and the still unresolved distribution of *R. australis* in Southeast Asia, this study aimed to assess its presence in continental Southeast Asia, specifically for Cambodia.

## 2. Materials and methods

The tick specimens used in the molecular analysis were collected from various locations across 21 provinces of Cambodia, including from cattle farms and household-level cattle rearing in rural and urban settings. Ticks were collected directly from cattle through visual inspection by trained technicians, who examined each animal for a total of 30 min. Ticks found on the cattle were carefully removed using fine-tipped forceps, by grasping the mouthparts and slowly pulling upward to ensure all mouthparts were properly detached from the skin, causing minimal discomfort to the animals. The collected ticks were transferred into 15 ml conical tubes containing 70 % ethanol and labeled according to host, habitat, and locations. In the laboratory, ticks were identified morphologically and sorted by species using species descriptions (Barker and Walker, 2014; Estrada-Pena et al., 2012; Walker et al., 2014; Yamaguti et al., 1971) and identification keys developed in-house by the Medical and Veterinary Entomology Unit at Institut Pasteur du Cambodge (IPC).

A subset of samples was randomly selected by species and location, resulting in a total of 95 individual ticks, morphologically identified as *R. australis* (*n* = 37), *R. microplus* s.l. (*n* = 39), and *Rhipicephalus* spp. nymphs (*n* = 19) and subjected to molecular analysis. DNA from ticks was extracted using a QIAmp DNA mini kit (Qiagen, Germany) with slight modifications to the protocol to incorporate non-destructive methods (Takhampunya et al., 2021). A small incision was made in the abdomen of the tick specimen with a surgical blade before the

addition 180 µL of buffer ATL and 20 µL of proteinase K. The ticks were then incubated overnight at 56 °C to allow sufficient enzymatic digestion of the tissues. The exoskeleton of the tick was transferred to a new 1.5 ml tube, washed several times in distilled water, and finally kept in 70 % ethanol for taxonomic study, while 200 µL of the supernatant was used to follow the subsequent steps of the DNA extraction kit, according to the manufacturer's protocol.

A fragment of *cox1* gene was amplified by PCR using a set of primer pairs LCO1490/HCO2198 (Folmer et al., 1994). Each 40 µL of PCR mix contained 4 µL of 10X PCR buffer B2 (Solis Biodyne, Estonia), 0.8 µL of each primer (10 µM), 0.4 µL of 20 mM dNTP mix, 5.6 µL of 25 mM mgCl₂, 0.4 µL of Hot firepol DNA polymerase (5U/µL) (Solis Biodyne, Estonia), 23 µL of nuclease-free water, and 5 µL of DNA template. PCR assays were conducted using a T100 thermal cycler (Biorad, USA) using protocol as follows: initial denaturation at 95 °C for 15 min; followed by 40 cycles of 95 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min; followed by a final extension of 72 °C for 10 min. The resulting amplicons (710 bp) were visualized using gel electrophoresis and were sent to Macrogen (Daejeon, South Korea) for purification and DNA sequencing.

The sequence reads were processed using Unipro UGENE version 50.0 (Okonechnikov et al., 2012) and the consensus sequences (GenBank accession number PQ838663-PQ838765, Table S2) were aligned with reference sequences retrieved from the GenBank database using ClustalW for multiple alignment. A maximum likelihood phylogenetic tree was constructed using the best-fit nucleotide substitution model with 1000 bootstrap replicates in IQ-TREE version 2.3.5 (Minh et al., 2020). The phylogenetic tree was visualized and edited using Interactive Tree of Life (iTOL) version 6.9.1 (Letunic and Bork, 2024).
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Maximum likelihood phylogenetic tree of *R. microplus* complex in Cambodia based on a fragment of the *cox1* gene. Text in red indicates sequences obtained from Genbank, while text in black represents sequences from this study. All *R. microplus* complex specimens, including those possessing *R. australis* characters (indicated by black dots), were grouped within *R. microplus* clade A.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** *Rhipicephalus microplus* clade A from Cambodia exhibit characteristics of *R. australis*, as indicated by yellow arrows. This includes the presence of abundant alloscutal setae (A) and setae posterior to the eyes (B) in female specimens, as well as the retroverted ventral spurs on palpal article 1 (C) in males.</figcaption></figure>
## 3. Results and discussion

During the nationwide tick sampling, a total of 5304 ticks belonging to the *R. microplus* complex were collected from 97 infested cattle out of 120 inspected. Among them, 1205 were morphologically identified as *R. australis*, 2239 as *R. microplus*, and 1860 immature stages. Both identified species were detected in nearly all sampled provinces, indicating a widespread distribution rather than a geographically clustered pattern (Fig. 1). However, despite these morphological classifications, *cox1* sequence analysis revealed that all ticks belonged to a single species, *R. microplus* (Fig. 2). This result is consistent with studies from Malaysia (*Kazim et al.*, 2022). Despite extensive nationwide sampling, the results indicate that *R. microplus* from Cambodia belongs exclusively to clade A, in contrast to studies from Malaysia and Thailand, where both clades A and C have been reported (Low *et al.*, 2015; Tantrawatpan *et al.*, 2022). However, the limited number of *cox1* sequences analyzed in this study raises the possibility that other clades, if present in very low numbers, may have gone undetected.

Some *R. microplus* from Cambodia possessed morphological characters of *R. australis*, particularly the presence of spurs on the ventral side of palpal article 1 in males, and the abundance of median alloscutal setae that clustered in four to six rows in females (Fig. 3). Same inconsistencies have been reported in Asia, central America and South Africa (*Guzmán et al.*, 2021; *Kaewmongkol et al.*, 2015; *Kazim et al.*, 2022; *Londt and Arthur*, 1975; *Roy et al.*, 2018). Most male specimens of *R. microplus* examined in a study from Thailand possess spurs on the ventral side of palpal article 1, a characteristic typically observed in *R. australis* males (*Kaewmongkol et al.*, 2015). Similar traits were also noted in specimens from Bangladesh, Myanmar, Pakistan, Cuba and South Africa (*Guzmán et al.*, 2021; *Londt and Arthur*, 1975; *Roy et al.*, 2018). Additionally, a female specimen of *R. microplus* from Myanmar exhibited abundant dorsal median alloscutal setae arranged in 4–6 rows, and females from Bangladesh, Myanmar, Pakistan displayed setae behind the eyes, both of which are characteristics of *R. australis* (*Roy et al.*, 2018). The high variation in these characters might be associated with the nature of this species complex, which follows a one-host life cycle that may facilitate cohabitation and potential cross-breeding, generating offspring with mixed characters (*Ali et al.*, 2016; *Berry*, 2017; *Kaewmongkol et al.*, 2015; *Kazim et al.*, 2022).

While the morphological characters and molecular data of *R. australis* appear consistent in specimens from Australia, Indonesia, and New Caledonia, this is not the case for Cambodia and other countries in Southeast Asia, where the presence was never supported by molecular data (*Estrada-Peña et al.*, 2012). The growing evidence of conflicting morphological characters may refute earlier claims of *R. australis* presence in the region and suggest that its actual range may not extend to continental Southeast Asia as previously thought. The findings of this study, along with similar observations elsewhere (*Guzmán et al.*, 2021; *Kaewmongkol et al.*, 2015; *Kazim et al.*, 2022; *Londt and Arthur*, 1975; *Roy et al.*, 2018), demonstrate that the morphological characters described by Estrada-Peña (*Estrada-Pena et al.*, 2012) cannot be used as the sole reference to distinguish *R. australis* from *R. microplus*. Until validated by molecular data, there is currently no evidence supporting the presence of *R. australis* in continental Southeast Asia.

### Ethical statement

The study was authorized and facilitated by the General Directorate of Animal Health and Production (GDAHP) within the Ministry of Agriculture, Forestry, and Fisheries of Cambodia, under permit No. 2981, issued on October 1, 2021.

### CRediT authorship contribution statement

Didot Budi Prasetyo: Writing – original draft, Visualization,

Methodology, Formal analysis, Data curation. **Sony Yean:** Writing – original draft, Methodology, Investigation, Data curation. **Sébastien Boyer:** Writing – review & editing, Supervision, Methodology, Funding acquisition, Conceptualization.

### Declaration of competing interest

The authors declare that they have no competing financial interests or personal relationships that could have influenced the work reported in this manuscript.

### Funding sources

This work was supported by The Solidarity Fund for Innovative Projects (FSPI), Embassy of France in Cambodia.

### Acknowledgements

The authors sincerely thank the personnel of the General Directorate of Animal Health and Production (GDAHP), Kingdom of Cambodia, as well as the local authorities for facilitating access and granting permission for animal inspections. Special thanks are also extended to the Medical and Veterinary Entomology Unit at IPC for their invaluable dedication to sample collection and identification, which significantly contributed to the success of this study.

### Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.ttbdis.2025.102478.

### Appendix. Supplementary material

Table S1. *Rhipicephalus microplus* *cox1* sequences generated in this study

### Data availability

All sequence data generated in this study have been deposited in GenBank and are publicly accessible under the accession numbers PQ838663-PQ838754, PQ838761, PQ838762, PQ838764, and PQ838765. Other data will be made available on request.

### References

Ali, A., Parizi, L.F., Ferreira, B.R., Vaz Junior, I., da, S., 2016. A revision of two distinct species of *Rhipicephalus*: *R. microplus* and *R. australis*. Ciênc. Rural 46, 1240–1248. https://doi.org/10.1590/0103-8478cr20151416.

Barker, S., Walker, A., 2014. Ticks of Australia. The species that infest domestic animals and humans. Zootaxa 3816, 1–144. https://doi.org/10.11646/zootaxa.3816.1.1.

Berry, C.M., 2017. Resolution of the Taxonomic Status of *Rhipicephalus (Boophilus)* *Microplus* (PhD). University of Glasgow.

Burger, T.D., Shao, R., Barker, S.C., 2014. Phylogenetic analysis of mitochondrial genome sequences indicates that the cattle tick, *rhipicephalus (Boophilus) microplus*, contains a cryptic species. Mol. Phylogenet. Evol. 76, 241–253. https://doi.org/10.1016/j.ympev.2014.03.017.

Chien, N., Linh, B., Van Tho, N., Hieu, D., Lan, N., 2016. Status of cattle ticks infection in yellow and dairy cows in Ba Vi District. In: Proc. Int. Conf. Agric. Dev. Context Int. Integr. Oppor. Chall. Hanoi Vietnam, pp. 115–119.

Estrada-Peña, A., Venzal, J.M., Nava, S., Mangold, A., Guglielmone, A.A., Labruna, M.B., de la Fuente, J., 2012. Reinstatement of *rhipicephalus (Boophilus) australis* (Acari: Ixodidae) with redescription of the adult and larval stages. J. Med. Entomol. 49, 794–802. https://doi.org/10.1603/me11223.

Folmer, O., Black, M.B., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol.

Guzmán, P.E.E., Cuétara, C.F., Argüelles, A.L.C., Castillo, A.F., Martínez, Y.G., Fernández, R.R., Afonso, Y.F., Soto, Y.B., Alfaro, Y.G., Méndez, L., García, A.D., García, M.P.E., Rodriguez-Mallon, A., 2021. Characterization of two Cuban colonies of *rhipicephalus microplus* ticks. Vet. Parasitol. Reg. Stud. Rep. 25, 100591. https://doi.org/10.1016/j.vprsr.2021.100591.
Hornok, S., Farkas, R., Duong, N.N., Kontschán, J., Takács, N., Keve, G., Pham, D.N., Dao, T.T.H., 2024. A morpho-phylogenetic update on ixodid ticks infesting cattle and buffalos in Vietnam, with three new species to the fauna and a checklist of all species indigenous to the country. Parasit. Vectors 17, 319. https://doi.org/10.1186/s13071-024-06384-5

Kaewmongkol, S., Kaewmongkol, G., Inthong, N., Lakkitjaroen, N., Sirinarumitr, T., Berry, C.M., Jonsson, N.N., Stich, R.W., Jittapalapong, S., 2015. Variation among Bm86 sequences in *Rhipicephalus (Boophilus) microplus* ticks collected from cattle across Thailand. Exp. Appl. Acarol. 66, 247–256. https://doi.org/10.1007/s10493-015-9897-0

Kazim, A.R., Low, V.L., Tappe, D., Houssaini, J., Heo, C.C., 2022. *Rhipicephalus annulatus*, *R. australis* or *R. microplus*? Discordance between morphological and genetic data among three cattle tick species. Exp. Appl. Acarol. 87, 119–131. https://doi.org/10.1007/s10493-022-00726-7

Letunic, I., Bork, P., 2024. Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Res 52, W78–W82. https://doi.org/10.1093/nar/gkae268.

Londt, J.G.H., Arthur, D.R., 1975. The structure and parasitic life cycle of *boophilus microplus* (Canestrini, 1888) in South Africa (Acarina: ixodidae). J. Entomol. Soc. South. Afr. 38, 321–340. https://doi.org/10.10520/AJA00128789_3653.

Low, V.L., Tay, S.T., Kho, K.L., Koh, F.X., Tan, T.K., Lim, Y.A.L., Ong, B.L., Panchadcharam, C., Norma-Rashid, Y., Sofian-Azirun, M., 2015. Molecular characterisation of the tick *rhipicephalus microplus* in Malaysia: new insights into the cryptic diversity and distinct genetic assemblages throughout the world. Parasit. Vectors 8, 341. https://doi.org/10.1186/s13071-015-0956-5

Minh, B.Q., Schmidt, H.A., Chernomor, O., Schrempf, D., Woodhams, M.D., von Haeseler, A., Lanfear, R., 2020. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol. Biol. Evol. 37, 1530–1534. https://doi.org/10.1093/molbev/msaa015

Okonechnikov, K., Golosova, O., Fursov, M., the UGENE team, 2012. Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics 28, 1166–1167. https://doi.org/10.1093/bioinformatics/bts091

Roy, B.C., Estrada-Peña, A., Krücken, J., Rehman, A., Nijhof, A.M., 2018. Morphological and phylogenetic analyses of *rhipicephalus microplus* ticks from Bangladesh, Pakistan and Myanmar. Ticks Tick-Borne Dis 9, 1069–1079. https://doi.org/10.1016/j.ttbdis.2018.03.035.

Takhampunya, R., Thaloengsok, S., Tippayachai, B., Promsathaporn, S., Leepitakrat, S., Gross, K., Davidson, S.A., 2021. Retrospective survey of *Borrelia* spp. From rodents and ticks in Thailand. J. Med. Entomol. 58, 1331–1344. https://doi.org/10.1093/jme/tjaa279.

Tan, L.P., Hamdan, R.H., Hassan, B.N.H., Reduan, M.F.H., Okene, I.A.-A., Loong, S.K., Khoo, J.J., Samsuddin, A.S., Lee, S.H., 2021. *Rhipicephalus* tick: a contextual review for Southeast Asia. Pathogens 10, 821. https://doi.org/10.3390/pathogens10070821.

Tantrawatpan, C., Vaisusuk, K., Chatan, W., Pilap, W., Suksavate, W., Andrews, R.H., Petney, T.N., Saijuntha, W., 2022. Genetic diversity and phylogenetic analyses of ixodid ticks infesting cattle in northeast Thailand: the discovery of *rhipicephalus microplus* clade C and the rarely detected *R. haemaphysaloides*. Exp. Appl. Acarol. 86, 535–548. https://doi.org/10.1007/s10493-022-00704-z.

Walker, A., Bouattour, A., J.-L. Camicas, Estrada-Peña, A., Horak, I., Latif, A., R.G. Pegram, P.M. Preston, 2014. Ticks of Domestic Animals in Africa: a guide to identification of species.

Yamaguti, N., Tipton, V., Keegan, H., Toshioka, S., 1971. Ticks of Japan, Korea, and the Ryukyu Islands. Brigh. Young Univ. Sci. Bull. Biol. Ser. 15.

Ybañez, A.P., Sivakumar, T., Ybañez, R.H.D., Ratilla, J.C., Perez, Z.O., Gabotero, S.R., Hakimi, H., Kawazu, S., Matsumoto, K., Yokoyama, N., Inokuma, H., 2013. First molecular characterization of *anaplasma marginale* in cattle and *rhipicephalus (Boophilus) microplus* ticks in Cebu, Philippines. J. Vet. Med. Sci. 75, 27–36. https://doi.org/10.1292/jvms.12-0268.