First in vitro feeding of the Australian marsupial tick, Ixodes hirsti Hassall, 1931, with preliminary microbiome profiling and observations on the nymphal morphology

Artificial tick feeding systems (ATFS) provide a valuable alternative to animal-based models for studying tick biology. Ixodes hirsti , an Australian tick species that parasitises marsupials, remains understudied due to challenges in laboratory maintenance. Here, we report the first successful in vitro feeding of I. hirsti larvae, provide preliminary microbiome profiles of unfed larvae and larvae recovered after artificial feeding and present the first molecularly confirmed morphological description of the nymphal stage. Field-collected engorged females of I. hirsti were allowed to oviposit under laboratory conditions. Hatched larvae were artificially fed on blood using silicone membranes supplemented with kangaroo hair and/or kangaroo hair extract. Microbiomes were characterised by 16S rRNA amplicon sequencing, while scanning electron microscopy (SEM) and sequencing of 16S rRNA and cox 1 genes were used for morphological and molecular characterisation of nymphs. Membranes treated with hair extract alone yielded the highest attachment rate (71%), whereas kangaroo hair-treated membranes produced superior feeding performance, with shorter time to engorgement (9.26 ± 1.00 days) and a higher engorgement weight (0.91 ± 0.01 mg). Exploratory microbiome profiling showed that fed larval pools had numerically lower microbial richness and evenness than unfed larval pools, although these differences were not significant. A total of 80 microbial taxa were shared between groups, whereas seven and 17 taxa were unique to fed and unfed larvae, respectively. Stenotrophomonas was more abundant in fed larval pools while Coxiella -like and Rickettsia -like endosymbionts were detected in both fed and unfed larvae. These findings demonstrate that ATFS can be adapted for wildlife-associated ticks with specialised host preferences and provide a practical framework for investigating the biology and microbial ecology of ticks.