Biology subjects

Slapeta, J.

Publications and source records attributed to Slapeta, J..

9 recordsLinked to original sources

bioRxiv2026

Transcriptomic and lipidomic profiles reveal higher thermal adaptability in a tropical versus a temperate isolate of Chromera velia

The apicomplexan alga Chromera velia is the closest known phototrophic relative of apicomplexan parasites (Sporozoa). Despite its scientific importance as a model for the evolution of parasitic Apicomplexa, only two strains are available in culture. The first isolate (SH), obtained from stony coral in the cold waters of Sydney Harbour, was used for the original species description. The second isolate (OTI) originates from tropical corals at One Tree Island in the Great Barrier Reef. In this study, we compare their growth, transcriptomic, and lipidomic profiles at 20{degrees}C and 26{degrees}C. Both strains grew faster at 26{degrees}C, yet they responded differently to higher temperature. In SH, warming downregulated nearly 85% of its thermally responsive genes, with motility-, cytoskeleton-, and lipid-associated genes most strongly affected, while at 26{degrees}C, SH retained higher expression of translation and nitrogen, amide, and peptide biosynthesis genes than OTI. OTI, by contrast, mounted a smaller, more balanced response and maintained stable expression of cilium, axoneme, and microtubule genes across both temperatures. The lipidomes of the two strains showed both shared and divergent responses. Both isolates sense and respond to increasing temperature in the same basic way, but OTI stabilizes its membranes, whereas SH redirects lipid synthesis from membrane lipids to storage lipids. OTI membranes were also constitutively less unsaturated than those of SH at both temperatures, consistent with a membrane already suited to warmer water that needs less remodeling. Our data show that two isolates of the same species, originating from different thermal environments, respond to elevated temperature through distinct transcriptomic and lipidomic programs. This suggests that the tropical OTI isolate is adapted to higher temperatures and demonstrates how reef-associated algae respond to rising ocean temperatures.

evolutionary biology↗

bioRxiv2025

Ambient temperature storage in DESS supports molecular studies of benzimidazole resistance from canine hookworm eggs

Emerging benzimidazole resistance in canine hookworms poses a growing concern for veterinary and public health. Molecular diagnostics targeting {beta}-tubulin gene mutations are essential for resistance surveillance but traditionally rely on refrigerated faecal samples. This study evaluates dimethyl sulfoxide, EDTA, and saturated NaCl (DESS) as a room-temperature preservation medium for canine faeces. Using ITS-2 rDNA and isotype-1 {beta}-tubulin gene (tubb-1) amplicon metabarcoding, we compared DNA integrity and diagnostic performance of DESS-preserved samples (4, 28, and 106 days) to refrigerated controls. No significant differences in PCR amplification or sequencing outcomes were observed. DESS enabled reliable detection of hookworm species and resistance-associated SNPs, including F167Y in tubb-1, with mutation frequencies consistent across treatments. Therefore, DESS can preserve samples from remote settings without cold chain logistics. Our findings validate DESS as a robust alternative for sample collection for molecular parasitology, facilitating expanded surveillance of anthelmintic resistance in field conditions. HighlightsO_LIDESS preserves canine faeces for DNA analysis without refrigeration. C_LIO_LIHookworm species and resistance SNPs detected reliably from DESS samples. C_LIO_LIPCR and NGS results from DESS samples match refrigerated controls. C_LIO_LIF167Y mutation found in A. caninum and U. stenocephala samples. C_LIO_LIDESS enables remote hookworm surveillance without cold chain logistics. C_LI

microbiology↗

bioRxiv2025

Characterisation of β-tubulin isotypes in Uncinaria stenocephala and implications for benzimidazole resistance in hookworms

Uncinaria stenocephala is a widespread hookworm of dogs across Europe, Canada, southern Australia, and other temperate regions, where it often outnumbers infections caused by Ancylostoma caninum. Although a putative {beta}-tubulin isotype-1 mutation associated with resistance has been detected in U. stenocephala, clinical resistance to benzimidazoles has not yet been confirmed. Benzimidazole resistance is primarily linked to single nucleotide polymorphisms (SNPs) in the {beta}-tubulin isotype-1 gene; however, the {beta}-tubulin genes of U. stenocephala have not been fully characterised. We aimed to identify {beta}-tubulin genes and confirm the coding sequences for key residues (Q134, F167, E198, and F200) in the {beta}-tubulin isotype-1 gene of the U. stenocephala genome. Two U. stenocephala specimens were subjected to Illumina sequencing, and species identity was confirmed through morphological and molecular analysis using ITS rDNA and cox-1 markers. Genome assembly revealed the presence of {beta}-tubulin isotype-1 (10 exons) and isotype-2 (9 exons), both homologous to {beta}-tubulins from other hookworms (A. caninum, A. ceylanicum, A. duodenale and Necator americanus). The {beta}-tubulin isotype-1 protein sequence of U. stenocephala contained two variable residues (S37Q and G441A) compared to other hookworm sequences. While the isotype-2 protein sequence was conserved among Ancylostoma species, U. stenocephala exhibited six distinct polymorphisms (E39D, T40S, N115S, L130I, A287S, T439G). The benzimidazole-susceptible residues (Q134, F167, E198, F200) were present in the {beta}-tubulin isotype-1 protein sequence. Characterisation of the complete coding regions of {beta}-tubulin isotypes 1 and 2 enables population-level screening for benzimidazole resistance-associated SNPs and provides a foundation for future epidemiological studies in U. stenocephala. Highlights{beta}-tubulin isotypes-1 and -2 were fully characterised in Uncinaria stenocephala Key benzimidazole-susceptible residues were confirmed in {beta}-tubulin isotype-1 {beta}-tubulin isotypes-1 and -2 enables SNP screening and resistance surveillance efforts O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/664546v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1598c5corg.highwire.dtl.DTLVardef@df5f0org.highwire.dtl.DTLVardef@8d8ec7org.highwire.dtl.DTLVardef@198b768_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2024

Population genomics reveals an ancient origin of heartworms in canids

Heartworms (Dirofilaria immitis) are parasitic nematodes that cause significant cardiopulmonary-associated morbidity and mortality in canids worldwide. The global spread of heartworms is believed to have occurred alongside the dispersal of domesticated dogs. To test this theory, we analysed the genomes of 127 specimens collected from mammalian carnivore hosts across four continents. Here we show distinct genetic differences between heartworms from different continents, indicating a more ancient dispersal in canid hosts than previously recognised. Using admixture analyses, we find an Asian origin for Australian heartworms, consistent with the arrival of dingoes thousands of years ago. Finally, the genetic relatedness between European and Central American heartworms suggests that modern dispersal, likely associated with human colonisation of the Americas by Europeans, occurred with domesticated dogs. This work sheds light on the population dynamics and deep evolutionary history of a globally widespread parasite of veterinary significance.

evolutionary biology↗

bioRxiv2024

Widespread occurrence of benzimidazole resistance SNPs in the canine hookworm, Ancylostoma caninum, in Australia

Canine hookworm (Ancylostoma caninum), a gastrointestinal nematode of domestic dogs, principally infects the small intestine of dogs and has the potential to cause zoonotic disease. In greyhounds and pet dogs in the USA, A. caninum has been shown to be resistant to multiple anthelmintics. We conducted a molecular survey of benzimidazole resistance in A. caninum from dogs at veterinary diagnostic centers in Australia and New Zealand. First, we implemented an internal transcribed spacer (ITS)-2 rDNA deep amplicon metabarcoding sequencing approach to ascertain the species of hookworms infecting dogs in the region. Then, we evaluated the frequency of the canonical F167Y and Q134H isotype-1 {beta}-tubulin mutations, which confer benzimidazole resistance, using the same sequencing approach. The most detected hookworm species in diagnostic samples was A. caninum (90%; 83/92); the related Northern hookworm (Uncinaria stenocephala) was identified in 11% (10/92) of the diagnostic samples. There was a single sample with coinfection by A. caninum and U. stenocephala. Both isotype-1 {beta}-tubulin mutations were present in A. caninum, 49% and 67% for Q134H and F167Y, respectively. Mutation F167Y in the isotype-1 {beta}-tubulin mutation was recorded in U. stenocephala for the first known time. Canonical benzimidazole resistance codons 198 and 200 mutations were absent. Egg hatch assays performed on a subset of the A. caninum samples showed significant correlation between 50% inhibitory concentration (IC50) to thiabendazole and F167Y, with an increased IC50 for samples with >75% F167Y mutation. We detected 14% of dogs with >75% F167Y mutation in A. caninum. Given that these samples were collected from dogs across various regions of Australia, the present study suggests that benzimidazole resistance in A. caninum is widespread. Therefore, to mitigate the risk of resistance selection and further spread, adoption of a risk assessment-based approach to limit unnecessary anthelmintic use should be a key consideration for future parasite control.

microbiology↗

bioRxiv2023

A mixed amplicon metabarcoding and sequencing approach for surveillance of drug resistance to levamisole and benzimidazole in Haemonchus spp.

Anthelmintic resistant parasitic nematodes present a significant threat to sustainable livestock production worldwide. The ability to detect the emergence of anthelmintic resistance at an early stage, and therefore determine which drugs remain most effective, is crucial for minimising production losses. Despite many years of research into the molecular basis of anthelmintic resistance, no molecular-based tools are commercially available for the diagnosis of resistance as it emerges in field settings. We described a mixed deep amplicon sequencing approach to determine the frequency of the levamisole (LEV) resistant single nucleotide polymorphism (SNP) within arc-8 exon 4 (S168T) in Haemonchus spp., coupled with benzimidazole (BZ) resistance SNPs within {beta}-tubulin isotype-1 and ITS-2 nemabiome. This constitutes the first multi-drug and multi-species molecular diagnostic developed for helminths of veterinary importance. Of the ovine, bovine, caprine and camelid Australian field isolates we tested, S168T was detected in the majority of Haemonchus spp. populations from sheep and goats, but rarely at a frequency greater than 16%; an arbitrary threshold we set based on whole genome sequencing of LEV resistant H. contortus GWBII. Overall, BZ resistance was far more prevalent in Haemonchus spp. than LEV resistance, confirming that LEV is still an important anthelmintic class for small ruminants in New South Wales. The mixed amplicon metabarcoding approach described herein, paves the way towards the use of large scale sequencing as a surveillance technology in the field, the results of which can be translated into evidence-based recommendations for the livestock sector.

microbiology↗

bioRxiv2023

Genome structure and population genomics of the canine heartworm Dirofilaria immitis

The heartworm, Dirofilaria immitis, is a filarial parasitic nematode responsible for significant morbidity and mortality in wild and domesticated canids. Resistance to macrocyclic lactone drug prevention represents a significant threat to parasite control and has prompted investigations to understand the genetic determinants of resistance. This study aimed to improve the genomic resources of D. immitis to enable a more precise understanding of how genetic variation is distributed within and between parasite populations worldwide, which will inform the likelihood and rate by which parasites, and in turn, resistant alleles, might spread. We have guided the scaffolding of a recently published genome assembly for D. immitis (ICBAS_JMDir_1.0) using the chromosomal-scale reference genomes of Brugia malayi and Onchocerca volvulus, resulting in an 89.5 Mb assembly composed of four autosomal- and one X-linked chromosomal-scale scaffolds representing 99.7% of the genome. Publicly available and new whole-genome sequencing data from 32 D. immitis samples from Australia, Italy and the USA were assessed using principal component analysis, nucleotide diversity (Pi) and absolute genetic divergence (Dxy) to characterise the global genetic structure and measure within- and between population diversity. These population genetic analyses revealed broad-scale genetic structure among globally diverse samples and differences in genetic diversity between populations; however, fine-scale subpopulation analysis was limited and biased by differences between sample types. Finally, we mapped SNPs previously associated with macrocyclic lactone resistance in the new genome assembly, revealing physical linkage of high-priority variants on chromosome 3, and determined their frequency in the studied populations. This new chromosomal assembly for D. immitis now allows for a more precise investigation of selection on genome-wide genetic variation and will enhance our understanding of parasite transmission and the spread of genetic variants responsible for resistance to treatment.

genomics↗

bioRxiv2022

Angie-LAMP for diagnosis of human eosinophilic meningitis using dog as proxy: A LAMP assay for Angiostrongylus cantonensis DNA in cerebrospinal fluid

BackgroundAngiostrongylus cantonensis (rat lungworm) is recognised as the leading cause of human eosinophilic meningitis, a serious condition observed when nematode larvae migrate through the CNS. Canine Neural Angiostrongyliasis (CNA) is the analogous disease in dogs. Both humans and dogs are accidental hosts, and rapid diagnosis is warranted. A highly sensitive PCR based assay is available but often not readily accessible in many jurisdictions. An alternative DNA amplification assay that would further improve the accessibility is needed. This study aimed to assess the diagnostic utility of a newly designed LAMP assay to detect DNA of globally distributed and invasive A. cantonensis and Angiostrongylus mackerrasae, the only other neurotropic Angiostrongylus species, which is native to Australia. Methodology/Principal FindingsCerebrospinal fluid (CSF) from dogs (2020-2022) with a presumptive diagnosis of A. cantonensis infection were received for confirmatory laboratory testing and processed for DNA isolation and ultrasensitive Angiostrongylus qPCR. A newly designed LAMP assay targeting AcanR3390 in a diagnostic laboratory setting was directly compared to the reference ultrasensitive qPCR for determination of presence of A. cantonensis DNA to aid the diagnosis of CNA. The LAMP assay (Angie-LAMP) allowed the sensitive detection of A. cantonensis DNA from archived DNA specimens (Kappa=0.81, 95%CI 0.69-0.92; n=93) and rapid single-step lysis of archived CSF samples (Kappa=0.77, 95%CI 0.59-0.94; n=52). Only A. cantonensis DNA was detected in canine CSF samples, and co-infection with A. mackerrasae using amplicon deep sequencing (ITS-2 rDNA) was not demonstrated. Both SYD.1 and AC13 haplotypes were detected using sequencing of partial cox1. Conclusions/SignificanceThe Angie-LAMP assay is a useful molecular tool for detecting Angiostrongylus DNA in CSF of dogs and performs comparably to laboratory Angiostrongylus qPCR. Adaptation of single-step sample lysis improved potential applicability for effective diagnosis of angiostrongyliasis in a clinical setting for dogs and by extension for humans. Authors summaryA potentially fatal disease, neural angiostrongyliasis, is caused by the rat lungworm (Angiostrongylus cantonensis). The parasite migrates into the spinal cord and brain of accidental hosts, such as humans and dogs, after ingestion of infective larvae. Recently, an ultrasensitive molecular assay which can detect tiny fragments of the parasites DNA was developed and has been used for confirmatory diagnosis. Although this assay outperforms previously developed assays, it requires clean DNA with specialised equipment in a laboratory setting. There is an urgent need for an alternative diagnostic method which is sensitive and portable, for deployment in the field and in the hospitals in remote areas or in low-income countries. The authors developed a fast and portable loop-mediated isothermal amplification (LAMP) assay that compares favourably to the ultra-sensitive PCR assay when tested using cerebrospinal fluid from dogs on the Australian east coast with presumptive neural angiostrongyliasis. Considering a One Health approach to diagnostics, this assay enables portable emergency diagnostics equally suitable to humans, dogs and wildlife. The newly developed assay will also enable water supplies to be screened, as well as crustaceans and molluscs used as potential food sources, for presence of the parasite.

microbiology↗

bioRxiv2022

Suspect macrocyclic lactone resistance cases with the canine heartworm (Dirofilaria immitis): mismatch between microfilarial suppression test and SNP results in Australia

Canine heartworm disease is a potentially deadly cardiopulmonary disease caused by the mosquito-borne filarial nematode Dirofilaria immitis. In Australia, the administration of macrocyclic lactone (ML) drugs has successfully reduced the prevalence of D. immitis infection. However, the recent re-emergence of D. immitis in dogs in Queensland, Australia and the identification of ML-resistant isolates in the USA poses an important question of whether ML-resistance has emerged in this parasite in Australia. The aim of this study was to utilise phenotypic and genotypic approaches to examine the sensitivity to ML drugs in D. immitis in Australia. To do this, we surveyed 45 dogs from Queensland and New South Wales across 3 years (2019-2022) for the presence of D. immitis infection using an antigen test, quantitative Modified Knotts test, and qPCR targeting both D. immitis and the D. immitis symbiont Wolbachia. A phenotype observed by utilising sequential quantification of microfilariae for 23/45 dogs was coupled with genetic testing of filtered microfilariae for SNPs previously associated with ML-resistance in isolates from the USA. Sixteen (16/45) dogs tested positive for D. immitis infection despite reportedly receiving rigorous heartworm prevention for 12 months prior to the study, according to the owners assessment. The phenotype and genotypic assays in this study did not unequivocally demonstrate the presence of ML-resistant D. immitis in Australia. Although the failure of 16 dogs to reduce microfilaremia by >90% after ML treatment was considered a suspect phenotype of ML-resistance, no genotypic evidence was discovered using the genetic SNP analysis. The traditional quantitative Modified Knotts test can be substituted by qPCR targeting D. immitis or associated Wolbachia endosymbiont DNA for a more rapid measurement of microfilariae levels. More definitive phenotypic evidence of resistance is critically needed before the usefulness of SNPs for the detection of ML-resistance in Australia can be properly assessed.

microbiology↗