BACKGROUND: Rats (Rattus spp.) invaded most of the world as stowaways including some that carried the rat lungworm, Angiostrongylus cantonensis, the cause of eosinophilic meningoencephalitis in humans and other warm-blooded animals. A high genetic diversity of A. cantonensis based on short mitochondrial DNA regions is reported from Southeast Asia. However, the identity of invasive A. cantonensis is known for only a minority of countries. The affordability of next-generation sequencing for characterisation of A. cantonensis genomes should enable new insights into rat lung worm invasion and parasite identification in experimental studies. METHODS: Genomic DNA from morphologically verified A. cantonensis (two laboratory-maintained strains and two field isolates) was sequenced using low coverage whole genome sequencing. The complete mitochondrial genome was assembled and compared to published A. cantonensis and Angiostrongylus malaysiensis sequences. To determine if the commonly sequenced partial cox1 can unequivocally identify A. cantonensis genetic lineages, the diversity of cox1 was re-evaluated in the context of the publicly available cox1 sequences and the entire mitochondrial genomes. Published experimental studies available in Web of Science were systematically reviewed to reveal published identities of A. cantonensis used in experimental studies. RESULTS: New A. cantonensis mitochondrial genomes from Sydney (Australia), Hawaii (USA), Canary Islands (Spain) and Fatu Hiva (French Polynesia), were assembled from next-generation sequencing data. Comparison of A. cantonensis mitochondrial genomes from outside of Southeast Asia showed low genetic diversity (0.02-1.03%) within a single lineage of A. cantonensis. Both cox1 and cox2 were considered the preferred markers for A. cantonensis haplotype identification. Systematic review revealed that unequivocal A. cantonensis identification of strains used in experimental studies is hindered by absence of their genetic and geographical identity. CONCLUSIONS: Low coverage whole genome sequencing provides data enabling standardised identification of A. cantonensis laboratory strains and field isolates. The phenotype of invasive A. cantonensis, such as the capacity to establish in new territories, has a strong genetic component, as the A. cantonensis found outside of the original endemic area are genetically uniform. It is imperative that the genotype of A. cantonensis strains maintained in laboratories and used in experimental studies is unequivocally characterised.

CEITEC VFU University of Veterinary and Pharmaceutical Sciences Brno Palackého třída 1946 1 612 42 Brno Czech Republic

Centre for Veterinary Education University of Sydney Sydney NSW 2006 Australia

Department of Pathology and Parasitology University of Veterinary and Pharmaceutical Sciences Brno Palackého třída 1946 1 612 42 Brno Czech Republic

Department of Pathology and Parasitology University of Veterinary and Pharmaceutical Sciences Brno Palackého třída 1946 1 612 42 Brno Czech Republic Institute of Parasitology Biology Center of the Czech Academy of Sciences Branišovská 1160 31 370 05 České Budějovice Czech Republic

Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias Universidad de La Laguna C Astrofisico F Sanchez s n Tenerife 38203 La Laguna Canary Islands Spain

Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias Universidad de La Laguna C Astrofisico F Sanchez s n Tenerife 38203 La Laguna Canary Islands Spain Department Obstetricia y Ginecología Pediatría Medicina Preventiva y Salud Pública Toxicología Medicina Legal y Forense y Parasitología Universidad de La Laguna 38203 San Cristóbal de La Laguna Canary Islands Spain

Marie Bashir Institute for infectious Diseases and Biosecurity University of Sydney Sydney NSW 2006 Australia

Sydney School of Veterinary Science University of Sydney Sydney NSW 2006 Australia

Sydney School of Veterinary Science University of Sydney Sydney NSW 2006 Australia Department of Pathology and Parasitology University of Veterinary and Pharmaceutical Sciences Brno Palackého třída 1946 1 612 42 Brno Czech Republic Institute of Vertebrate Biology Czech Academy of Sciences Květná 8 603 65 Brno Czech Republic

USDA APHIS WS National Wildlife Research Center Hawaii Field Station PO Box 10880 Hilo HI 96721 USA Manaaki Whenua Landcare Research PO Box 69040 Lincoln 7608 New Zealand

Westmead Clinical School University of Sydney Sydney NSW 2145 Australia

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