The greater wax moth, Galleria mellonella, has quietly become one of the most useful small workhorses in infection biology. Its larvae are cheap to rear, produce large numbers of offspring, complete their life cycle quickly, and can be infected and treated without the ethical approvals required for vertebrate experiments. Over the past decade they have been used to model host-pathogen interactions, characterize innate immune responses, and test the efficacy of new drugs. Yet despite this popularity, a fundamental piece of genomic information has been missing: nobody had systematically mapped where transcription begins across the moth’s genome. A new study in iScience fills that gap, presenting the first draft atlas of transcription start sites for G. mellonella and doing so under conditions that matter for disease research, namely fungal infection and antifungal drug treatment.
The research team, led by Imad Abugessaisa of the RIKEN Center for Integrative Medical Sciences together with colleagues including Wendy van de Sande of Erasmus MC, built their atlas using a technique called LQ-ssCAGE, or low-quantity single-strand cap analysis of gene expression. CAGE-based methods capture the 5-prime capped ends of RNA molecules, which mark the exact positions where RNA polymerase II initiated transcription. Because the method both identifies these transcription start sites and quantifies how abundantly each one is used, it provides a two-in-one view of promoter activity and gene expression. The researchers prepared 72 LQ-ssCAGE libraries and, in parallel, 72 standard RNA-seq libraries from the same samples, using the RNA-seq data as an independent quality check on the CAGE measurements.
The experimental design was carefully constructed to mimic a real infection and its treatment. Half of the larvae were infected with Madurella mycetomatis, a high-priority fungal pathogen on the World Health Organization’s fungal priority list and the most common cause of eumycetoma, a neglected tropical disease characterized by tumorous lesions in subcutaneous tissue. The infected and uninfected larvae were then divided into three groups: one treated with solvent only, one with the antifungal drug itraconazole, and one with ravuconazole, which was recently assessed in the first clinical trial for eumycetoma. Samples were collected at four time points, 6, 30, 72, and 168 hours, with three biological replicates per condition, producing a rich time-course dataset of promoter activity during infection and therapy.
Sequencing yielded roughly 53.3 million cap-trapped 5-prime reads, which the team mapped to the current reference genome assembly, CSIRO_AGI_GalMel_v1, a high-quality assembly scoring 99.3 percent on the lepidoptera BUSCO benchmark and containing 15,138 genes and pseudogenes. Quality control was thorough. Gene body coverage showed the expected strong 5-prime enrichment for CAGE libraries and uniform coverage for RNA-seq libraries, sequencing depth was consistent across samples with no outlier batches, and pairwise correlation analysis between normalized RNA-seq and CAGE counts showed strong positive Spearman correlations, confirming that the two independent assays produced reproducible expression profiles.
From the raw data, the researchers identified 1,219,124 individual CAGE transcription start sites, which they filtered down to 332,601 biologically relevant positions. Clustering these signals produced 40,200 tag clusters, from which they derived two tiers of transcription start site peaks: a permissive set of 27,942 peaks detected in at least four samples, and a robust set of 5,437 peaks detected in all samples. They also identified 728 bidirectional clusters, the molecular signature of active enhancers, which after filtering to non-exonic intronic and intergenic locations yielded 141 candidate active enhancers. Users of the atlas can therefore choose between the permissive and robust annotations depending on how conservative their analyses need to be.
The validation evidence for these calls is strong. Among all CAGE peaks, 32.1 percent overlapped annotated 5-prime untranslated regions, and more strikingly, 80.27 percent of all CAGE tags fell within 5-prime UTRs, exactly where active promoters should sit. Genome-wide enrichment analysis showed that the identified peaks were strongly and specifically enriched at annotated promoters of both protein-coding and non-coding RNA genes, while intronic and intergenic regions were depleted. Dinucleotide frequency analysis at the dominant start sites revealed the characteristic initiator sequence bias expected at insect RNA polymerase II promoters, and the atlas-derived annotations displayed a markedly stronger core promoter signature, including TATA box and GC patch signals, than the existing NCBI reference annotation, demonstrating a genuine improvement in transcription start site resolution.
The atlas also revealed the two canonical shapes of animal promoters. Using the interquartile range of tag clustering as a measure of initiation precision, the team classified 4,746 broad promoters, where transcription starts at many scattered positions, and 1,679 sharp promoters, where initiation is concentrated at a single precise site. As expected from earlier CAGE studies in mammals and flies, sharp promoters carried a stronger TATA box positioned roughly 30 bases upstream of the start site, while broad promoters were GC-rich, consistent with CpG island-associated architecture. Most genes, about 6,899, used a single dominant start site, whereas roughly 2,300 genes used multiple sites, and 71.1 percent of promoter-associated genes were linked to more than one tag cluster, echoing the widespread alternative promoter usage documented in Drosophila.
Perhaps the most clinically intriguing result concerns what did and did not change during infection. RNA-seq confirmed differentially expressed genes between infected and healthy larvae, consistent with the team’s earlier work on iron regulatory pathways during M. mycetomatis grain development. However, no differentially expressed genes were detected between infected larvae and infected larvae treated with itraconazole or ravuconazole. The authors note that this mirrors recent clinical observations in which neither drug showed measurable activity during the first six months of eumycetoma treatment, with persistent fungal viability and no reduction in lesion size or plasma beta-1,3-D-glucan levels. Differential transcription start site usage analysis did flag a handful of genes, including a cystinosin homolog and a peroxidase-like isoform, whose promoter selection shifted between healthy and infected samples, hinting that infection may add a layer of transcriptional regulation beyond simple changes in gene abundance.
Using expression correlations between nearby elements, the team predicted physical interactions between enhancers and promoters, finding that 682 of 37,320 transcription start sites interacted with 156 of 218 active enhancers. Interacting start sites were expressed at significantly higher levels than non-interacting ones, and the analysis also uncovered super-enhancers, clusters of closely spaced bidirectional enhancers. De novo motif discovery on the top 5 percent of expressed start sites pointed to RFX family transcription factors, with the RFX1 ortholog showing 7.1-fold enrichment, a factor conserved across animals and known in flies as a key regulator of ciliary and sensory neuron genes. The authors are candid about limitations: only about 700,000 reads per sample were generated, and saturation analysis suggests the depth was sufficient for highly expressed promoters but likely underestimated weak enhancers. Even so, with all data deposited in NCBI GEO and Mendeley Data, and the annotations browsable in the ZENBU genome browser, this first draft start site atlas gives the growing G. mellonella research community a foundation for gene regulation studies comparable to the reference resources long available for human and mouse.
Subject of Research: Genome-wide mapping of transcription start sites in Galleria mellonella larvae during fungal infection and antifungal treatment
Article Title: Genome-wide mapping of the Galleria mellonella transcription start sites during fungal infection and treatment
Article References: Abugessaisa, I., Konings, M., Manabe, R.-I., Tagami, M., Severin, J., Hasegawa, A., Kawashima, T., Kinoshita, H., Noma, S., Takahashi, C., Verbon, A., Okazaki, Y., van de Sande, W. W., & Kasukawa, T. (2026). Genome-wide mapping of the Galleria mellonella transcription start sites during fungal infection and treatment. iScience, 29(10), Article 117675. https://doi.org/10.1016/j.isci.2026.117675
Image Credits: AI Generated
DOI: 10.1016/j.isci.2026.117675
Keywords: Galleria mellonella, transcription start sites, CAGE, Madurella mycetomatis, eumycetoma, promoters, enhancers, gene regulation, itraconazole, ravuconazole, genome annotation, infection model
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Juliet Wilcox. (October 3, 2026). Wax Moth Larvae Yield First Genome-Wide Map of Transcription Start Sites During Fungal Infection. Scienmag. https://scienmag.com/wax-moth-larvae-yield-first-genome-wide-map-of-transcription-start-sites-during-fungal-infection/
Juliet Wilcox. “Wax Moth Larvae Yield First Genome-Wide Map of Transcription Start Sites During Fungal Infection.” Scienmag, 3 October 2026, https://scienmag.com/wax-moth-larvae-yield-first-genome-wide-map-of-transcription-start-sites-during-fungal-infection/. Accessed 3 October 2026.
Juliet Wilcox. “Wax Moth Larvae Yield First Genome-Wide Map of Transcription Start Sites During Fungal Infection.” Scienmag. October 3, 2026. https://scienmag.com/wax-moth-larvae-yield-first-genome-wide-map-of-transcription-start-sites-during-fungal-infection/
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