Biology subjects

Gulia-Nuss, M.

Publications and source records attributed to Gulia-Nuss, M..

6 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↗

bioRxiv2025

A cytochrome P450 G subfamily member, CYP4G35, is highly expressed in antennae and modulates olfactory response in Aedes aegypti mosquitoes.

The cytochrome P450 enzymes of the CYP4G subfamily are some of the most enigmatic insect P450s. The dipterans with sequenced genomes have two CYP4G paralogs. In Drosophila melanogaster, CYP4G1 is highly expressed in the oenocytes and catalyzes the last enzymatic step in the biosynthesis of cuticular hydrocarbons. In contrast, CYP4G15 is expressed in the brain glial cells, but its function is unknown. The Aedes aegypti genome encodes two CYP4Gs: CYP4G36 (ortholog of DmCYP4G1) and CYP4G35 (ortholog of DmCYP4G15). Here, we show that CYP4G35 is highly expressed in mosquito antennae, and the RNAi knockdown of CYP4G35 results in delayed host-seeking. Ae. aegypti CYP4G knockout lines confirmed delayed host-seeking behavior in CYP4G35 knockout females. Proteomics analysis of CYP4G35 KO females also corroborates the physiological findings and shows upregulation of proteins related to olfaction and other CYP4Gs to compensate for the lack of CYP4G35. Immunohistochemistry and in situ hybridization were used to localize CYP4G35 and demonstrated its expression in the sensilla lymph of the antennae and the tip of the proboscis. CYP4G35 and CYP4G36 fusion proteins with cytochrome P450 reductase demonstrated that, unlike CYP4G36, CYP4G35 lacks an oxidative decarbonylase function. Together, our data support a novel function of CYP4G35 in modulating olfactory response.

molecular biology↗

bioRxiv2025

Developmental system drift in the patterning of the arthropod tarsus

The current understanding of proximodistal axis patterning in arthropod legs is grounded in insect models. The paradigm for appendage evolution in this phylum is that the gene regulatory network responsible for leg subdivision and patterning is broadly conserved. Recent surveys of these genes have suggested that chelicerate exemplars exhibit divergent appendage patterning dynamics, though functional data remain limited. One salient mismatch in expression occurs in homologs of the homeobox gene clawless. In insects, clawless is expressed in the distalmost leg territory, specifying the claw-bearing pretarsus. In the harvestman, Phalangium opilio, clawless occupies a broad tarsal domain early in development, localizing later to the metatarsus-tarsus boundary, suggestive of a tarsal patterning function. Here, we tested the function of harvestman clawless using RNAi. Unlike insects, we show that clawless knockdown results in disrupted tarsal growth and patterning of its proximal segmental boundary, with no effect on the claw. Truncation of the tarsus is associated with defective tarsomere formation. We additionally surveyed clawless homologs in exemplars of chelicerate diversity, which suggests that the tarsal-patterning function for clawless was likely present in the chelicerate common ancestor. These results, alongside available expression data, suggest panarthropod appendage patterning exhibits numerous cases of developmental system drift.

evolutionary biology↗

bioRxiv2024

A novel expression domain of extradenticle underlies the evolutionary developmental origin of the chelicerate patella

Neofunctionalization of duplicated gene copies is thought to be an important process underlying the origin of evolutionary novelty and provides an elegant mechanism for the origin of new phenotypic traits. One putative case where a new gene copy has been linked to a novel morphological trait is the origin of the arachnid patella, a taxonomically restricted leg segment. In spiders, the origin of this segment has been linked to the origin of the paralog dachshund-2, suggesting that a new gene facilitated the expression of a new trait. However, various arachnid groups that possess patellae do not have a copy of dachshund-2, disfavoring the direct link between gene origin and trait origin. We investigated the developmental genetic basis for patellar patterning in the harvestman Phalangium opilio, which lacks dachshund-2. Here, we show that the harvestman patella is established by a novel expression domain of the transcription factor extradenticle. Leveraging this definition of patellar identity, we surveyed targeted groups across chelicerate phylogeny to assess when this trait evolved. We show that a patellar homolog is present in Pycnogonida (sea spiders) and various arachnid orders, suggesting a single origin of the patella in the ancestor of Chelicerata. A potential loss of the patella is observed in Ixodida. Our results suggest that the modification of an ancient gene, rather than the neofunctionalization of a new gene copy, underlies the origin of the patella. Broadly, this work underscores the value of comparative data and broad taxonomic sampling when testing hypotheses in evolutionary developmental biology.

evolutionary biology↗

bioRxiv2023

Validation of a heat-inducible Ixodes scapularis HSP70 promoter and developing a tick-specific 3xP3 promoter sequence in ISE6 cells

Ixodes scapularis is an important vector of many pathogens, including the causative agent of Lyme disease, tick-borne encephalitis, and anaplasmosis. The study of gene function in I. scapularis and other ticks has been hampered by the lack of genetic tools, such as an inducible promoter to permit temporal control over transgenes encoding protein or double-stranded RNA expression. Studies of vector-pathogen relationships would also benefit from the capability to activate anti-pathogen genes at different times during pathogen infection and dissemination. We have characterized an intergenic sequence upstream of the heat shock protein 70 (HSP70) gene that can drive Renilla luciferase expression and mCherry fluorescence in the I. scapularis cell line ISE6. In another construct, we replaced the Drosophila melanogaster minimal HSP70 promoter in the synthetic 3xP3 promoter with a minimal portion of the I. scapularis HSP70 promoter and generated an I. scapularis specific 3xP3 (Is3xP3) promoter. Both promoter constructs, IsHSP70 and Is3xP3, allow for heat-inducible expression of mCherry fluorescence in ISE6 cells with an approximately 10-fold increase in the percentage of fluorescent positive cells upon exposure to a 2 h heat shock. These promoters described here will be valuable tools for gene function studies and temporal control of gene expression, including anti-pathogen genes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/569248v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@b5ae25org.highwire.dtl.DTLVardef@1bc267borg.highwire.dtl.DTLVardef@1826baborg.highwire.dtl.DTLVardef@16acf4e_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2018

Chicago and Dovetail Hi-C proximity ligation yield chromosome length scaffolds of Ixodes scapularis genome

A high-quality genome sequence is essential for understanding an organism on molecular level. However, the larger genomes with substantial repetitive sequences are challenging to assemble with the sequencing technologies. Hi-C technique is changing the genome architecture landscape by providing links across a variety of length scales, spanning even whole chromosomes. Ixodes scapularis haploid genome is 2.1 gbp and the current assembly consists of 369,495 scaffolds representing 57% of the genome. The fragmented genome poses challenges with functional gene analysis and an improved assembly is needed. We therefore used the Hi C technique to achieve chromosomal level assembly of tick genome. With Chicago and Dovetail Hi C assemblies, we were able to achieve 28 >10Mb sequences that correspond to 28 chromosomes in I. scapularis.

genomics↗