Molecular characterisation of Australasian Ixodiphagus (Hymenoptera; Encyrtidae; Encyrtinae) reveals unexpected diversity and a potential novel host switch.

Ticks are important medical and veterinary parasites that represent a substantial health threat to humans, companion animals, and livestock. Ixodiphagus wasps (Hymenoptera; Encyrtidae) are known endoparasitoids of ixodid (hard) and argasid (soft) ticks, with potential utility as natural biocontrol agents. Two species, Ixodiphagus brunneus and Ixodiphagus mysorensis, are previously recorded from Australia, however, the genus lacks formal revisionary work in Australia, and the validity and host ranges of these species remain uncertain. This work aimed to investigate the diversity of Ixodiphagus in Australasia and provide a molecular data resource for future work on these understudied endoparasitoids. We extracted DNA from archival Ixodiphagus specimens from Australian and New Zealand insect collections and performed high-throughput sequencing which resulted in complete or mostly complete mitochondrial genome sequences from 11 specimens, including I. brunneus, Ixodiphagus taiaroaensis, and a novel Ixodiphagus sp. reared from Rhipicephalus linnaei from Townsville, Australia. In addition, approximately 70% of the genome of the Wolbachia endosymbiont of I. brunneus was recovered. Finally, we screened 178 recently collected pooled tick samples from southern New South Wales, Australia, for Ixodiphagus spp. using 28S rRNA and cytochrome c oxidase subunit 1(COI) gene PCR, and recovered 14 positive samples. Phylogenetic analysis of Australasian Ixodiphagus spp. based on 28S rRNA and complete mitochondrial genome sequences determined that members of the Australasian fauna are distinct from Ixodiphagus hookeri (the only other Ixodiphagus species for which genetic data exists), and that at least two distinct species are present in Australia; I. brunneus identified from Ixodes holocyclus and Haemaphysalis bancrofti ticks, and an uncharacterised Ixodiphagus sp. found in Rhipicephalus linnaei ticks from northern Queensland. Furthermore, there was substantial genetic diversity at the 28S rRNA loci among I. brunneus samples, which may represent normal genetic variability or a secondary cryptic species. The molecular data generated here represents the first known for the genus Ixodiphagus in Australasia, doubling that of the world fauna, and provides the first known complete mitochondrial genomes for these important tick parasitoids.

Tick-borne piroplasms and trypanosomes incidentally detected in eastern grey kangaroos (Macropus giganteus) during a mortality and morbidity event in southern New South Wales, Australia.

Tick-borne haemoparasites, including piroplasms and trypanosomes, are almost ubiquitous in Australian wildlife, with some associated with health impacts to individual animals and declining wildlife populations. An array of ecologically distinct piroplasm and trypanosome species occur throughout Australia although many of these species and their sylvatic ecologies are poorly characterised. Between May 2022 and October 2023, an anecdotally reported localised eastern grey kangaroo (Macropus giganteus) morbidity/mortality event occurred in coastal southern New South Wales, Australia, characterised by animals presenting with blindness, emaciation, lethargy, ataxia, and astasia. Here we used molecular techniques to identify tick-borne piroplasms (Babesia and Theileria) and trypanosomes in affected animals. Blood (n = 89) and liver (n = 19) samples were collected after the humane euthanasia of wild animals due to welfare concerns, and brief notes on the animal's health were recorded. In total, 20 (22.5%) animals were infected with tick-borne haemoparasites, including a novel Theileria sp. nov. (14, 15.7%), Babesia macropus (2, 2.2%), Trypanosoma gilletti (5, 5.6%), and Trypanosoma vegrandis (1, 1.1%). Liver samples were also screened for Wallal and Warego viruses due to animals' blindness, but were negative. This is the first report of T. gilletti and T. vegrandis in eastern grey kangaroos, although they have been previously reported in high numbers in ticks which commonly parasites this host. The novel Theileria sp. was previously reported in questing Ixodes holocyclus and in ticks from an opportunistically collected eastern grey kangaroo and red-necked wallaby (Notamacropus rufogriseus). However, we show for the first time this Theileria sp. can occur widely in eastern grey kangaroos. Ultimately, this small study did not intend, and is not able to draw inference regarding the pathogenicity of these haemoparasites to eastern grey kangaroos and it is likely that other factors, such as chronic Phalaris grass toxicity, had a role in this localised mortality/morbidity event.

Characterisation and comparative genomics of three new Varanus-associated Borrelia spp. from Indonesia and Australia.

BACKGROUND: Borrelia are important disease-causing tick- and louse-borne spirochaetes than can infect a wide variety of vertebrates, including humans and reptiles. Reptile-associated (REP) Borrelia, once considered a peculiarity, are now recognised as a distinct and important evolutionary lineage, and are increasingly being discovered worldwide in association with novel hosts. Numerous novel Borrelia spp. associated with monitor lizards (Varanus spp.) have been recently identified throughout the Indo-Pacific region; however, there is a lack of genomic data on these Borrelia. METHODS: We used metagenomic techniques to sequence almost complete genomes of novel Borrelia spp. from Varanus varius and Varanus giganteus from Australia, and used long- and short-read technologies to sequence the complete genomes of two strains of a novel Borrelia sp. previously isolated from ticks infesting Varanus salvator from Indonesia. We investigated intra- and interspecies genomic diversity, including plasmid diversity and relatedness, among Varanus-associated Borrelia and other available REP Borrelia and, based on 712 whole genome orthologues, produced the most complete phylogenetic analysis, to the best of our knowledge, of REP Borrelia to date. RESULTS: The genomic architecture of Varanus-associated Borrelia spp. is similar to that of Borrelia spp. that cause relapsing fever (RF), and includes a highly conserved megaplasmid and numerous smaller linear and circular plasmids that lack structural consistency between species. Analysis of PF32 and PF57/62 plasmid partitioning genes indicated that REP Borrelia plasmids fall into at least six distinct plasmid families, some of which are related to previously defined Borrelia plasmid families, whereas the others appear to be unique. REP Borrelia contain immunogenic variable major proteins that are homologous to those found in Borrelia spp. that cause RF, although they are limited in copy number and variability and have low sequence identities to RF variable major proteins. Phylogenetic analyses based on single marker genes and 712 single copy orthologs also definitively demonstrated the monophyly of REP Borrelia as a unique lineage. CONCLUSIONS: In this work we present four new genomes from three novel Borrelia, and thus double the number of REP Borrelia genomes publicly available. The genomic characterisation of these Borrelia clearly demonstrates their distinctiveness as species, and we propose the names Borrelia salvatorii, 'Candidatus Borrelia undatumii', and 'Candidatus Borrelia rubricentralis' for them.

Metatranscriptomic profiling reveals diverse tick-borne bacteria, protozoans and viruses in ticks and wildlife from Australia.

Tick-borne zoonoses are emerging globally due to changes in climate and land use. While the zoonotic threats associated with ticks are well studied elsewhere, in Australia, the diversity of potentially zoonotic agents carried by ticks and their significance to human and animal health is not sufficiently understood. To this end, we used untargeted metatranscriptomics to audit the prokaryotic, eukaryotic and viral biomes of questing ticks and wildlife blood samples from two urban and rural sites in New South Wales, Australia. Ixodes holocyclus and Haemaphysalis bancrofti were the main tick species collected, and blood samples from Rattus rattus, Rattus fuscipes, Perameles nasuta and Trichosurus vulpecula were also collected and screened for tick-borne microorganisms using metatranscriptomics followed by conventional targeted PCR to identify important microbial taxa to the species level. Our analyses identified 32 unique tick-borne taxa, including 10 novel putative species. Overall, a wide range of tick-borne microorganisms were found in questing ticks including haemoprotozoa such as Babesia, Theileria, Hepatozoon and Trypanosoma spp., bacteria such as Borrelia, Rickettsia, Ehrlichia, Neoehrlichia and Anaplasma spp., and numerous viral taxa including Reoviridiae (including two coltiviruses) and a novel Flaviviridae-like jingmenvirus. Of note, a novel hard tick-borne relapsing fever Borrelia sp. was identified in questing H. bancrofti ticks which is closely related to, but distinct from, cervid-associated Borrelia spp. found throughout Asia. Notably, all tick-borne microorganisms were phylogenetically unique compared to their relatives found outside Australia, and no foreign tick-borne human pathogens such as Borrelia burgdorferi s.l. or Babesia microti were found. This work adds to the growing literature demonstrating that Australian ticks harbour a unique and endemic microbial fauna, including potentially zoonotic agents which should be further studied to determine their relative risk to human and animal health.

Novel Hendra Virus Variant Detected by Sentinel Surveillance of Horses in Australia.

We identified and isolated a novel Hendra virus (HeV) variant not detected by routine testing from a horse in Queensland, Australia, that died from acute illness with signs consistent with HeV infection. Using whole-genome sequencing and phylogenetic analysis, we determined the variant had ≈83% nt identity with prototypic HeV. In silico and in vitro comparisons of the receptor-binding protein with prototypic HeV support that the human monoclonal antibody m102.4 used for postexposure prophylaxis and current equine vaccine will be effective against this variant. An updated quantitative PCR developed for routine surveillance resulted in subsequent case detection. Genetic sequence consistency with virus detected in grey-headed flying foxes suggests the variant circulates at least among this species. Studies are needed to determine infection kinetics, pathogenicity, reservoir-species associations, viral-host coevolution, and spillover dynamics for this virus. Surveillance and biosecurity practices should be updated to acknowledge HeV spillover risk across all regions frequented by flying foxes.

Diurnal variation in expired breath volatiles in malaria-infected and healthy volunteers.

We previously showed that thioether levels in the exhaled breath volatiles of volunteers undergoing controlled human malaria infection (CHMI) with P. falciparum increase as infection progresses. In this study, we show that thioethers have diurnal cyclical increasing patterns and their levels are significantly higher in P. falciparum CHMI volunteers compared to those of healthy volunteers. The synchronized cycle and elevation of thioethers were not present in P. vivax-infection, therefore it is likely that the thioethers are associated with unique factors in the pathology of P. falciparum. Moreover, we found that time-of-day of breath collection is important to accurately predict (98%) P. falciparum-infection. Critically, this was achieved when the disease was asymptomatic and parasitemia was below the level detectable by microscopy. Although these findings are encouraging, they show limitations because of the limited and logistically difficult diagnostic window and its utility to P. falciparum malaria only. We looked for new biomarkers in the breath of P. vivax CHMI volunteers and found that a set of terpenes increase significantly over the course of the malaria infection. The accuracy of predicting P. vivax using breath terpenes was up to 91%. Moreover, some of the terpenes were also found in the breath of P. falciparum CHMI volunteers (accuracy up to 93.5%). The results suggest that terpenes might represent better biomarkers than thioethers to predict malaria as they were not subject to malaria pathogens diurnal changes.

Advantages of substituting bioluminescence for fluorescence in a resonance energy transfer-based periplasmic binding protein biosensor.

A genetically encoded maltose biosensor was constructed, comprising maltose binding protein (MBP) flanked by a green fluorescent protein (GFP(2)) at the N-terminus and a Renilla luciferase variant (RLuc2) at the C-terminus. This Bioluminescence resonance energy transfer(2) (BRET(2)) system showed a 30% increase in the BRET ratio upon maltose binding, compared with a 10% increase with an equivalent fluorescence resonance energy transfer (FRET) biosensor. BRET(2) provides a better matched Förster distance to the known separation of the N and C termini of MBP than FRET. The sensor responded to maltose and maltotriose and the response was completely abolished by introduction of a single point mutation in the BRET(2) tagged MBP protein. The half maximal effective concentration (EC(50)) was 0.37 µM for maltose and the response was linear over almost three log units ranging from 10nM to 3.16 µM maltose for the BRET(2) system compared to an EC(50) of 2.3 µM and a linear response ranging from 0.3 µM to 21.1 µM for the equivalent FRET-based biosensor. The biosensor's estimate of maltose in beer matched that of a commercial enzyme-linked assay but was quicker and more precise, demonstrating its applicability to real-world samples. A similar BRET(2)-based transduction scheme approach would likely be applicable to other binding proteins that have a "venus-fly-trap" mechanism.

Effect of enhanced Renilla luciferase and fluorescent protein variants on the Förster distance of Bioluminescence resonance energy transfer (BRET).

Bioluminescence resonance energy transfer (BRET) is an important tool for monitoring macromolecular interactions and is useful as a transduction technique for biosensor development. Förster distance (R(0)), the intermolecular separation characterized by 50% of the maximum possible energy transfer, is a critical BRET parameter. R(0) provides a means of linking measured changes in BRET ratio to a physical dimension scale and allows estimation of the range of distances that can be measured by any donor-acceptor pair. The sensitivity of BRET assays has recently been improved by introduction of new BRET components, RLuc2, RLuc8 and Venus with improved quantum yields, stability and brightness. We determined R(0) for BRET(1) systems incorporating novel RLuc variants RLuc2 or RLuc8, in combination with Venus, as 5.68 or 5.55 nm respectively. These values were approximately 25% higher than the R(0) of the original BRET(1) system. R(0) for BRET(2) systems combining green fluorescent proteins (GFP(2)) with RLuc2 or RLuc8 variants was 7.67 or 8.15 nm, i.e. only 2-9% greater than the original BRET(2) system despite being ~30-fold brighter.

Comparison of enhanced bioluminescence energy transfer donors for protease biosensors.

Bioluminescence energy transfer (BRET) is a powerful tool for the study of protein-protein interactions and conformational changes within proteins. We directly compared two recently developed variants of Renilla luciferase (RLuc), RLuc2 and RLuc8, as BRET donors using an in vitro thrombin assay. The comparison was carried out by placing a thrombin-specific cleavage sequence between the donor luciferase and a green fluorescent protein (GFP(2)) acceptor. Substitution of native RLuc with the RLuc mutants, RLuc2 and 8, in a BRET(2) fusion protein increased the light output by a factor of ~10. Substitution of native RLuc with either of the RLuc mutants resulted in a decrease in BRET(2) ratio by a factor of ~2 when BRET(2) components were separated by the thrombin cleavage sequence. BRET(2) ratios changed by factors of 18.8±1.2 and 18.2±0.4 for GFP(2)-RG-RLuc2 and GFP(2)-RG-RLuc8 fusion proteins, respectively, on thrombin cleavage compared to 28.8±0.20 for GFP(2)-RG-RLuc. The detection limits for thrombin were 0.23 and 0.26 nM for RLuc2 and RLuc8 BRET(2) systems, respectively, and 15 pM for GFP(2)-RG-RLuc. However, overall, the mutant BRET systems remain more sensitive than FRET and brighter than standard BRET(2).

Behavioural and genetic evidence for C. elegans' ability to detect volatile chemicals associated with explosives.

BACKGROUND: Automated standoff detection and classification of explosives based on their characteristic vapours would be highly desirable. Biologically derived odorant receptors have potential as the explosive recognition element in novel biosensors. Caenorhabditis elegans' genome contains over 1,000 uncharacterised candidate chemosensory receptors. It was not known whether any of these respond to volatile chemicals derived from or associated with explosives. METHODOLOGY/PRINCIPAL FINDINGS: We assayed C. elegans for chemotactic responses to chemical vapours of explosives and compounds associated with explosives. C. elegans failed to respond to many of the explosive materials themselves but showed strong chemotaxis with a number of compounds associated with commercial or homemade explosives. Genetic mutant strains were used to identify the likely neuronal location of a putative receptor responding to cyclohexanone, which is a contaminant of some compounded explosives, and to identify the specific transduction pathway involved. Upper limits on the sensitivity of the nematode were calculated. A sensory adaptation protocol was used to estimate the receptive range of the receptor. CONCLUSIONS/SIGNIFICANCE: The results suggest that C. elegans may be a convenient source of highly sensitive, narrowly tuned receptors to detect a range of explosive-associated volatiles.