Biology subjects

Kittelmann, S.

Publications and source records attributed to Kittelmann, S..

7 recordsLinked to original sources

bioRxiv2026

ExTaSy: A swappable CRISPR platform for endogenous tagging in Drosophila melanogaster

Gene tagging enables functional analysis of proteins in vivo. Currently existing technologies in Drosophila suffer from drawbacks including limited flexibility, copy number variation, and/or imprecise expression. To address this, we have developed the Exchangeable Tagging System (ExTaSy), a CRISPR/Cas9-based platform which introduces a 3XHA tag into the endogenous gene locus. Importantly, the 3XHA can be subsequently exchanged for other tags using fly crosses, making the platform highly versatile and accessible. Simultaneously, an excisable transgenic marker allows virtually scarless locus modification. Here, we report successful tagging of 55 different loci, which shows that ExTaSy can be used to tag genes across the Drosophila genome and demonstrate its versatility for functional studies. We also developed a software that automates guide RNA and homology arm design, aiding efficient synthesis of transgenesis constructs. This novel technology will significantly improve our ability to visualize and manipulate proteins using various applications in vivo while maintaining endogenous expression levels and genetic background.

genetics↗

bioRxiv2025

Transgenerational dynamics of gut microbiota in black soldier fly larvae (Hermetia illucens) reared on a novel substrate

Understanding the transgenerational dynamics of gut microbiota in black soldier fly larvae (BSFL) is essential for optimizing their performance on novel waste substrates in industrial settings. In this study, a wild-type BSF population was divided into six sub-lines and reared over four generations: one on standard chicken feed (CF), five on a novel diet (WIL), four of which were additionally subjected to directional selection for larval size. Despite their shared genetic origin, sub-lines exhibited divergent trajectories in larval weight and gut bacterial composition. Larval weight increased up to the second (G2) or third generation (G3) but declined sharply at generation four (G4) across all lines. Parent-offspring regressions indicated low narrow-sense heritability and minimal genetic contribution to larval weight. Gut microbiota analysis revealed that early developmental stages were most sensitive to generational shifts, with G3 to G4 transitions showing the strongest shifts in microbial communities. Notably, certain taxa such as Bacillus and Paenibacillus, involved in cellulose degradation, peaked in G2 to G3 but declined at G4, whereas Klebsiella, associated with immune modulation, became more abundant. These trends suggested a shift from growth-associated to digestion-oriented microbial strategies under prolonged dietary stress. However, the absence of universally beneficial taxa and the stochastic emergence of distinct microbial patterns across sub-lines highlighted the plastic and lineage-specific nature of the BSFL gut microbiota. This study emphasizes the critical need for maintaining large, genetically and microbially diverse populations in BSF breeding programs to support long-term stability and avoid performance decline when adapting to novel or suboptimal substrates. ImportanceThe black soldier fly is increasingly used worldwide to convert organic waste into high-value protein, but the long-term stability of its gut microbiota on novel diets remains poorly understood. This study examined transgenerational changes in larval gut microbial communities from a single genetic population reared on a novel diet, with and without selection for larval size. Despite a shared genetic origin, sub-lines developed distinct microbiota and growth patterns, with early developmental stages showing the greatest sensitivity to generational microbial shifts. Initial increases in certain bacterial groups were followed by community restructuring by the fourth generation, indicating a dynamic but unstable microbial response to prolonged dietary stress. These findings highlight the importance of preserving microbial and genetic diversity when breeding black soldier flies for industrial use. Understanding how microbiota respond to selection and diet across generations is essential for sustaining performance and ensuring resilience in large-scale black soldier fly production systems.

microbiology↗

bioRxiv2025

Evolution under Domestication: Genetic differentiation in black soldier fly (Hermetia illucens) populations subjected to recent selective breeding

The black soldier fly (BSF; Hermetia illucens) is widely utilized in commercial and research applications for waste bioconversion and sustainable protein production. However, prolonged captivity and artificial selection can shape genetic diversity, potentially influencing adaptability and long-term population stability. This study examined how recent selective breeding, genetic drift, and relaxed selection have influenced genetic differentiation in BSF populations over a short timeframe. Using mitochondrial cytochrome oxidase I (CO1) and genome-wide RAD sequencing, population structure, heterozygosity, and selection signatures across eleven BSF populations, including selectively bred, wild-derived, and commercial strains were analysed. Results revealed that rapid genetic shifts have occurred within selectively bred populations (LA to LE) over [~]5 years, driven by artificial selection, subsequent relaxation, and environmental adaptation. The decline in effective population size (Ne) observed post-COVID-19 suggests recent bottlenecks, which may have further contributed to genetic drift and differentiation. While domesticated populations exhibited reduced genetic diversity and signs of inbreeding, wild-type population under short captivity retained higher heterozygosity. Genome-wide analyses further identified adaptive divergence among populations, with balancing selection and selective sweeps shaping genetic variation. Notably, despite shared ancestry, genetic differentiation persisted in selectively bred populations, reinforcing that selection and environmental pressures continue to influence their genomic landscape even after targeted selection was relaxed. These findings underscore the need to monitor genetic diversity in BSF breeding programs to maintain adaptability, enhance resilience, and mitigate risks from artificial selection and population collapse.

evolutionary biology↗

bioRxiv2020

Phylotype-Level Characterization of Complex Lactobacilli Communities Using a High-Throughput, High-Resolution Phenylalanyl-tRNA Synthetase (pheS) Gene Amplicon Sequencing Approach

The lactobacilli to date encompass more than 270 closely related species that were recently re-classified into 26 genera. Because of their relevance to industry, there is a need to distinguish between closely related, yet metabolically and regulatory distinct species, e.g., during monitoring of biotechnological processes or screening of samples of unknown composition. Current available methods, such as shotgun metagenomics or rRNA-based amplicon sequencing have significant limitations (high cost, low resolution, etc.). Here, we generated a lactobacilli phylogeny based on phenylalanyl-tRNA synthetase (pheS) genes and, from it, developed a high-resolution taxonomic framework which allows for comprehensive and confident characterization of lactobacilli community diversity and structure at the species-level. This framework is based on a total of 445 pheS gene sequences, including sequences of 277 validly described species and subspecies (out of a total of 283, coverage of 98%). It allows differentiation between 263 lactobacilli species-level clades out of a total of 273 validly described species (including the proposed species L. timonensis) and a further two subspecies. The methodology was validated through next-generation sequencing of mock communities. At a sequencing depth of [~]30,000 sequences, the minimum level of detection was approximately 0.02 pg per l DNA (equalling approximately 10 genome copies per {micro}l template DNA). The pheS approach along with parallel sequencing of partial 16S rRNA genes revealed a considerable lactobacilli diversity and distinct community structures across a broad range of samples from different environmental niches. This novel complementary approach may be applicable to industry and academia alike. IMPORTANCESpecies within the former genera Lactobacillus and Pediococcus have been studied extensively at the genomic level. To accommodate for their exceptional functional diversity, the over 270 species were recently re-classified into 26 distinct genera. Despite their relevance to both academia and industry, methods that allow detailed exploration of their ecology are still limited by low resolution, high cost or copy number variations. The approach described here makes use of a single copy marker gene which outperforms other markers with regards to species-level resolution and availability of reference sequences (98% coverage). The tool was validated against a mock community and used to address lactobacilli diversity and community structure in various environmental matrices. Such analyses can now be performed at broader scale to assess and monitor lactobacilli community assembly, structure and function at the species (in some cases even at sub-species) level across a wide range of academic and commercial applications.

microbiology↗

bioRxiv2020

Characterisation of the role and regulation of Ultrabithorax in sculpting fine-scale leg morphology

Hox genes are expressed during embryogenesis and determine the regional identity of animal bodies along the antero-posterior axis. However, they also function post-embryonically to sculpt fine-scale morphology. To better understand how Hox genes are integrated into post-embryonic gene regulatory networks, we further analysed the role and regulation of Ultrabithorax (Ubx) during leg development in Drosophila melanogaster. Ubx regulates several aspects of bristle and trichome patterning on the femurs of the second (T2) and third (T3) leg pairs. We found that repression of trichomes in the proximal posterior region of the T2 femur by Ubx is likely mediated by activation of the expression of microRNA-92a by this Hox protein. Furthermore, we identified a novel enhancer of Ubx that recapitulates the temporal and regional activity of this gene in T2 and T3 legs. We then used transcription factor binding motif analysis in regions of accessible chromatin in T2 leg cells to predict and functionally test transcription factors that may regulate the Ubx leg enhancer. We also tested the role of the Ubx co-factors Homothorax (Hth) and Extradenticle (Exd) in T2 and T3 femurs. We found several transcription factors that may act upstream or in concert with Ubx to modulate trichome patterning along the proximo-distal axis of developing femurs and that the repression of trichomes also requires Hth and Exd. Taken together our results provide insights into how Ubx is integrated into a postembryonic gene regulatory network to determine fine-scale leg morphology.

developmental biology↗

bioRxiv2019

A restriction enzyme reduced representation sequencing approach for low-cost, high-throughput metagenome profiling

Microbial community profiles have been associated with a variety of traits, including methane emissions in livestock, however, these profiles can be difficult and expensive to obtain for thousands of samples. The objective of this work was to develop a low-cost, high-throughput approach to capture the diversity of the rumen microbiome. Restriction enzyme reduced representation sequencing (RE-RRS) using ApeKI or PstI, and two bioinformatic pipelines (reference-based and reference-free) were compared to 16S rRNA gene sequencing using repeated samples collected two weeks apart from 118 sheep that were phenotypically extreme (60 high and 58 low) for methane emitted per kg dry matter intake (n=236). DNA was extracted from freeze-dried rumen samples using a phenol chloroform and bead-beating protocol prior to sequencing. The resulting sequences were used to investigate the repeatability of the rumen microbial community profiles, the effect of host genetics, laboratory and analytical method, and the genetic and phenotypic correlations with methane production. The results suggested that the best method was PstI RE-RRS analyzed with the reference-free approach via a correspondence analysis, with estimates for repeatability of 0.62{+/-}0.06, heritability 0.31{+/-}0.29, and genetic and phenotypic correlation with methane emissions of 0.88{+/-}0.25 and 0.64{+/-}0.05 respectively for the first component of correspondence analysis. The reference-free approach assigned 62.0{+/-}5.7% of reads to common 65 bp tags, much higher than the reference-based approach of 6.8{+/-}1.8% of reads assigned. Sensitivity studies suggested approximately 2000 samples could be sequenced in a single lane on an Illumina HiSeq 2500, therefore the current work of 118 samples/lane and future proposed 384 samples/lane are well within that threshold. Our approach is now being used to investigate host factors affecting the rumen and its association with a variety of production and environmental traits. With minor adaptations, our approach could be used to obtain microbial profiles from other metagenomic samples.

microbiology↗

bioRxiv2017

Gene regulatory network architecture in different developmental contexts influences the genetic basis of morphological evolution

Convergent phenotypic evolution is often caused by recurrent changes at particular nodes in the underlying gene regulatory networks (GRNs). The genes at such evolutionary hotspots are thought to maximally affect the phenotype with minimal pleiotropic consequences. This has led to the suggestion that if a GRN is understood in sufficient detail, the path of evolution may be predictable. The repeated evolutionary loss of larval trichomes among Drosophila species is caused by the loss of shavenbaby (svb) expression. svb is also required for development of leg trichomes, but the evolutionary gain of trichomes in the naked valley on T2 femurs in Drosophila melanogaster is caused by the loss of microRNA-92a (miR-92a) expression rather than changes in svb. We compared the expression and function of components between the larval and leg trichome GRNs to investigate why the genetic basis of trichome pattern evolution differs in these developmental contexts. We found key differences between the two networks in both the genes employed, and in the regulation and function of common genes. These differences in the GRNs reveal why mutations in svb are unlikely to contribute to leg trichome evolution and how instead miR-92a represents the key evolutionary switch in this context. Our work shows that variability in GRNs across different developmental contexts, as well as whether a morphological feature is lost versus gained, influence the nodes at which a GRN evolves to cause morphological change. Therefore, our findings have important implications for understanding the pathways and predictability of evolution.\n\nAuthor SummaryA major goal of biology is to identify the genetic cause of organismal diversity. Convergent evolution of traits is often caused by changes in the same genes - evolutionary hotspots. shavenbaby is a hotspot for larval trichome loss in Drosophila, however microRNA-92a underlies the gain of leg trichomes. To understand this difference in the genetics of phenotypic evolution, we compared the expression and function of genes in the underlying regulatory networks. We found that the pathway of evolution is influenced by differences in gene regulatory network architecture in different developmental contexts, as well as by whether a trait is lost or gained. Therefore, hotspots in one context may not readily evolve in a different context. This has important implications for understanding the genetic basis of phenotypic change and the predictability of evolution.

evolutionary biology↗