The mitochondrial genome of the pentastome parasite Raillietiella orientalis Hett, 1915 (Raillietiellida; Raillietiellidae) with notes on its phylogenetic position

Abstract In this study we sequenced and annotated the complete mitochondrial genome of the invasive reptile parasite Raillietiella orientalis using Illumina DNA sequencing. The length of the mitogenome was 15,320 bp and had a GC content of 33.1%. The mitogenome contained 13 protein-coding genes, two ribosomal RNA genes, and 22 tRNA genes, the order of which was diagnostic of Pancrustacean mitogenomes. A phylogenetic tree constructed from the 13 protein-coding genes of R. orientalis and 26 other Pancrustacean mitogenomes supported the placement of R. orientalis as part of the monophyletic subclass Pentastomida within the Maxillopoda and sister to the subclass Branchiura.

Pentastomes are endoparasitic crustaceans that largely parasitize the lungs of carnivorous reptiles (Pare 2008). The pentastome Raillietiella orientalis (Hett 1915) (Figure 1) is native to southeast Asia where it infects a diverse assemblage of herpetofauna including snakes and large lizards as definitive hosts, anurans and small lizards as secondary intermediate hosts, and invertebrates as primary intermediate hosts (Walden et al. 2020;Fieldsend et al. 2021;Palmisano et al. 2022). The abundant and diverse set of intermediate and definitive hosts infected by R. orientalis facilitates its establishment and spread in novel systems. There are currently two known invasion fronts of R. orientalis -Australia and North Americawhere infections in native snake populations are severe and often fatal (Kelehear et al. 2014;Farrell et al. 2019;Bogan et al. 2022). The origins of R. orientalis invasions are poorly understood, however, introduced populations of Burmese pythons in Florida may have been the initial source of parasite spillover in North America (Miller et al. 2018).
The taxonomy and systematics of the Pentastomida have long been disputed. Originally, this monophyletic clade was classified as its own phylum, but was subsequently repositioned to a subclass of Crustaceans (Lavrov et al. 2004). Over 130 species of pentastomids are recognized and distributed amongst two orders: Cephalobaenida and Porocephalida. Two genera in the Porocephalida, Armillifer and Linguatula, occur as zoonoses and have published mitogenomes (Lavrov et al. 2004;Naude et al. 2018). Raillietiella orientalis belongs to the Cephalobaenida for which no genomic resources are available. To help resolve their phylogenetic relations with other pentastomids, and to provide information that might be useful to researchers studying the conservation problems this invasive species poses, we present the mitochondrial genome of R. orientalis.
A deceased female southern black racer (Coluber constrictor) was discovered on the road of a residential neighborhood in Seminole County, Florida, USA (28.6349263 N, 81.2773628 W). The snake had R. orientalis adults exiting out of her glottis and buccal cavity at the time of encounter. The snake and pentastomes were collected and stored at À20 C. The snake was dissected and 61 adult R. orientalis, including 33 females (mean length ¼ 55 mm) and 28 males (mean length ¼ 12 mm), were removed from the mouth, trachea, lungs, and body cavity of the snake. All specimens were preserved in 70% ethanol following dissection.
Genomic DNA was extracted from a single adult female R. orientalis specimen using the Quick-DNA Miniprep kit (Zymo Research, Irvine, CA) according to the manufacturer's guidelines. The entire pentastome was used (17.3 mg), thus was not archived, but the DNA is available under voucher ID Ror_ cc_003 and additional specimens from the same host are archived at the University of Central Florida. A total of 1.4 mg of DNA was submitted to Novogene (Oxford, UK) for Illumina DNA sequencing. The genomic DNA was randomly fragmented, end-repaired, A-tailed, and ligated with Illumina adapters. The resulting fragments were PCR-amplified, size selected, and sequenced on a NovaSeq 6000 (Illumina, San Diego, CA) using paired-end 150-bp chemistry. A total of 354,419,038 paired reads were generated totaling 106.3 gigabases.
The   in R. orientalis was consistent with that described for Pancrustaceans (Boore 1999). The order of tRNA and rRNA genes included several rearrangements, which are a common feature of crustacean mitogenomes (Tan et al. 2019;Castellucci et al. 2022). Importantly, the R. orientalis mitogenome shares the position of tRNA-Leu2 (uaa) between COX1 and COX2 with other pentastome mitogenomes (Lavrov et al. 2004), which is considered the derived state in Pancrustaceans (Boore and Brown 1998;Boore 1999).

Ethics statement
The host snake (C. constrictor) was deceased at the time of collection and thus exempt from ethical handling approval or permissions. The host nor parasite were endangered or protected species and required no additional permits for their collection and use in Florida.

Disclosure statement
No potential conflict of interest was reported by the author(s).