Biology subjects

Wudarski, J.

Publications and source records attributed to Wudarski, J..

4 recordsLinked to original sources

bioRxiv2025

Copy Number Variation-Based Molecular Sexing of Ixodes scapularis and Rhipicephalus microplus Immature Stages Using qPCR and ddPCR Approaches

Accurate sex identification of immature ticks is essential for understanding sex-specific ecological dynamics, pathogen transmission, and reproductive biology. However, tick larvae and nymphs lack morphological sexual dimorphisms, limiting the studies. Here, we report the development and validation of copy number variation (CNV)-based molecular sexing approach for two important hard tick species, Ixodes scapularis, a major public health vector of human pathogens, and Rhipicephalus microplus, a pest responsible for significant economic losses in cattle industry. Using newly published chromosomal-level genome assemblies and whole-genome resequencing data, we identified female-enriched CNVs in two genes, calcium/calmodulin-dependent 3,5-cyclic nucleotide phosphodiesterase 1A-like (NPD) and rap guanine nucleotide exchange factor 2-like (RAPGEF2), and developed SYBR-Green based and probe-based qPCR, and droplet digital PCR (ddPCR) assays using DNA extracted non-destructively from a single leg, preserving ticks for continued feeding, molting, and behavioral analysis. In I. scapularis, we were able to assign the sex of 87% of nymphs based on results from at least two molecular tests, and these assignments were confirmed when the nymphs later molted into adults. In R. microplus, two qPCR assays revealed clear, sex-linked differences in gene copy number, despite the species having an XX: XO sex system. Together, these findings indicate that CNV-based markers are a reliable and widely applicable method for sex determination in immature ticks. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/695610v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@14616c5org.highwire.dtl.DTLVardef@80e758org.highwire.dtl.DTLVardef@d46484org.highwire.dtl.DTLVardef@1324e5_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Heat stress drives transcription of LTR retrotransposons in the regenerative flatworm Macrostomum lignano

The evolutionary arms race between transposable elements (TEs) and their hosts contributes to genomic complexity. As TEs mobilization is deleterious for individual cells and organisms, their activity is restricted. During stress, TEs can be reactivated; however, the exact mechanisms vary. We discovered that in the flatworm Macrostomum lignano, LTR retrotransposons hijack the heat shock response pathway to boost their transcription at elevated temperatures. While it has been well-described in cruciferous plants, this is the first report of this mechanism in animal LTR retrotransposons. Our results suggest a convergent evolution of the heat stress response in LTR retrotransposons from animals and plants.

genomics↗

bioRxiv2018

Influence of temperature on the development, reproduction and regeneration in the flatworm model organism Macrostomum lignano

The free-living marine flatworm Macrostomum lignano is a powerful model organism to study mechanisms of regeneration and stem cell regulation due to its convenient combination of biological and experimental properties, including the availability of transgenesis methods, which is unique among flatworm models. However, due to its relatively recent introduction in research, there are still many biological aspects of the animal that are not known. One of such questions is the influence of the culturing temperature on Macrostomum biology. Here we systematically investigated how different culturing temperatures affect the development time, reproduction rate, regeneration, heat shock response, and gene knockdown efficiency by RNA interference in M. lignano. We used marker transgenic lines of the flatworm to accurately measure the regeneration endpoint and to establish the stress response threshold for temperature shock. We found that compared to the culturing temperature of 20{degrees}C commonly used for M. lignano, elevated temperatures of 25{degrees}C-30{degrees}C substantially speed-up the development and regeneration time and increase reproduction rate without detectable negative consequences for the animal, while temperatures above 30{degrees}C elicit a heat shock response.\n\nWe show that altering the temperature conditions can be used to shorten the time required to establish M. lignano cultures, store important lines and optimize the microinjection procedures for transgenesis. Our findings will help to optimize the design of experiments in M. lignano and thus facilitate future research in this model organism.

zoology↗

bioRxiv2017

A platform for efficient transgenesis in Macrostomum lignano, a flatworm model organism for stem cell research

Regeneration-capable flatworms are informative research models to study the mechanisms of stem cell regulation, regeneration and tissue patterning. However, the lack of transgenesis methods significantly hampers their wider use. Here we report development of a transgenesis method for Macrostomum lignano, a basal flatworm with excellent regeneration capacity. We demonstrate that microinjection of DNA constructs into fertilized one-cell stage eggs, followed by a low dose of irradiation, frequently results in random integration of the transgene in the genome and its stable transmission through the germline. To facilitate selection of promoter regions for transgenic reporters, we assembled and annotated the M. lignano genome, including genome-wide mapping of transcription start regions, and showed its utility by generating multiple stable transgenic lines expressing fluorescent proteins under several tissue-specific promoters. The reported transgenesis method and annotated genome sequence will permit sophisticated genetic studies on stem cells and regeneration using M. lignano as a model organism.

bioengineering↗