Biology subjects

Papathanos, P. A.

Publications and source records attributed to Papathanos, P. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Cholesterol links blood feeding to mosquito development and reproduction

Mosquitoes undergo major metabolic remodeling as they transition from aquatic larvae to terrestrial, blood-feeding adults, yet the biochemical principles supporting these life cycle transitions remain poorly understood1. Early metabolic studies established that mosquitoes are sterol auxotrophs2,3; however, the extent to which this auxotrophy influences developmental progression and stage transitions remains unknown. Here, using sterol-defined culture systems, we uncover a central and stage-specific role for sterol metabolism in mosquitoes. We show that eggs of multiple mosquito species, from Anopheles gambiae (Giles 1902; hereafter A. gambiae) to Aedes species in both wild and laboratory settings, are enriched in cholesterol consistent with maternal provisioning. In the Asian tiger mosquito Aedes albopictus (Skuse, 1894; hereafter Ae. albopictus), egg cholesterol supports early larval development, after which larvae depend on the acquisition of dietary sterols to complete development. Like cholesterol, dietary plant- and fungal-derived sterols support late larval development; however, their utilization is associated with the accumulation of the intermediate sterol desmosterol. Using biochemical assays, we showed that desmosterol is converted to cholesterol through the activity of the Ae. albopictus DHCR-24 enzyme. This metabolic axis of desmosterol-to-cholesterol conversion supports late larval development when mosquitoes rely on plant- and fungal-derived sterols for development. dhcr-24 expression is upregulated during the developmental window of dietary sterol acquisition and is selectively induced by its substrate desmosterol, revealing a diet-encoded regulatory mechanism that coordinates metabolic conversion capacity. Extending the analysis to adulthood, we developed a sterol-defined artificial blood-feeding system that enables precise manipulation of dietary sterols in reproductive females and show that cholesterol availability in the diet of females is an essential metabolic driver of egg laying. These findings reveal a developmentally coordinated sterol-use strategy in the life cycle of Aedes mosquitoes, identifying sterol utilization as a central physiological axis linking development, blood feeding, and reproduction, and revealing a sterol-dependent metabolic vulnerability in mosquitoes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/725405v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@13c7843org.highwire.dtl.DTLVardef@fb9203org.highwire.dtl.DTLVardef@2f0db8org.highwire.dtl.DTLVardef@12db355_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

The Y chromosome gene draupnir reveals constraints on engineering Y-linked sex-ratio distorters in malaria mosquitoes

Y chromosome sex distorters offer a powerful strategy for mosquito population suppression, but their implementation is constrained by the transcriptional silencing of Y-linked transgenes during spermatogenesis. To investigate how endogenous Y genes evade this repression, we characterized draupnir (formerly YG5), a multicopy Y-linked gene of Anopheles gambiae encoding a Zip3-like meiotic protein. Comparative genomic and phylogenetic analyses revealed that draupnir originated through duplication of the autosomal paralog skirnir, itself derived from the ancestral recombination factor vilya, followed by amplification into a tandem array on the Y chromosome. We show that draupnir is actively transcribed during meiosis, making it the only known Y-linked gene expressed during mosquito spermatogenesis. To test whether its regulatory region is sufficient to confer sperm-specific expression from the Y chromosome, we cloned the draupnir promoter to drive expression of an X-chromosome shredder. When inserted on an autosome, the construct drove meiotic expression and strong sex-ratio distortion in the progeny of transgenic males. In contrast, the same construct inserted on the Y chromosome was transcriptionally silent and produced balanced sex ratios. These results demonstrate that draupnir expression depends on its native genomic context within a multicopy Y-linked array and that its promoter alone cannot overcome transcription silencing. Our findings reveal a fundamental constraint on Y-chromosome-based genetic control strategies and point to future approaches for enabling transcription from otherwise repressed sex chromosomes.

genetics↗

bioRxiv2026

Spermatogenic context controls outcomes of engineered sex distortion in malaria mosquitoes

Sex-ratio distortion systems are promising genetic tools for mosquito population control. Two strategies have been proposed: prezygotic elimination of X-bearing sperm by X-shredding, which can drive invasive Y-chromosome transmission when sex distorters are Y-linked, and postzygotic daughter killing through disruption of X-linked haploinsufficient genes, a self-limiting approach known as X-poisoning. Previous attempts to implement X-poisoning in the malaria mosquito Anopheles gambiae unexpectedly produced prezygotic distortion, with sex bias arising from loss of X-bearing sperm rather than daughter lethality. Here we use a split CRISPR-Cas9 system to systematically compare sex-ratio distortion outcomes across germline Cas9 drivers and X-linked target genes. Meiotic X-chromosome targeting induced preferential Y-chromosome transmission regardless of target identity, function, or number of sgRNA target sites. In contrast, shifting Cas9 expression to earlier spermatogenic stages altered outcomes dramatically: targeting X-linked ribosomal protein genes caused severe developmental or reproductive toxicity, whereas targeting the haplolethal muscle gene wupA produced daughter-specific post-embryonic lethality, with the majority of surviving females emerging flightless. Tracking offspring using a Y-linked fluorescent marker confirmed that sex chromosome segregation remained unbiased, with female mortality accumulating progressively from the first larval instar, reaching near-complete lethality by adulthood. These results demonstrate that the timing of Cas9 expression during spermatogenesis, rather than target gene identity alone, determines the outcome of X-chromosome targeting in malaria mosquitoes, and establish the conditions required for genuine X-poisoning. Identification of wupA as an effective X-poisoning target provides a solid foundation for the future development of self-limiting Y-linked sex-ratio distortion systems for malaria vector control.

genetics↗

bioRxiv2025

YOLito: A generalizable model for automated mosquito detection

Understanding mosquito behavior is key to advancing research in ecology, evolution, and disease control, yet most behavioral assays rely on human-dependent methods, such as real-time observation or manual frame-by-frame annotation, limiting throughput and reproducibility. We present YOLito, a domain-generalized AI model for automated mosquito detection and behavioral quantification. Built on the Ultralytics YOLO framework and enhanced with Slicing-Aided Hyper Inference (SAHI), YOLito accurately detects multiple mosquitoes across diverse backgrounds and imaging conditions. Trained on a globally assembled dataset of 38,547 annotated images from 35 experimental setups across six laboratories and three public datasets, YOLito achieved high performance on unseen data (precision = 0.95; recall = 0.91) and generalized across mosquito species (Aedes, Anopheles, Culex) and assay types, including blood-feeding, sugar-feeding, and oviposition. By automating behavioral scoring, YOLito transforms traditional assays into scalable and reproducible experimental platforms. The accompanying open-source toolkit enables high-throughput extraction of metrics such as visit frequency, duration, and distance traveled, providing a standardized and extensible framework that bridges computer vision and vector biology.

animal behavior and cognition↗

bioRxiv2025

3D-Feedy: An open and portable membrane feeder for mosquito research

Female mosquitoes must feed on blood to produce eggs, a natural behavior that also drives disease transmission. Vector biology research and the development of novel control tools depend on reliable methods for blood feeding of laboratory-reared mosquitoes. Artificial membrane feeders have provided a practical alternative to live animals for decades. However, commercial systems are typically expensive, difficult to repair and are tethered to external water baths or bench-top power supplies. Their limited capacity, usually only a few cages at once, further restricts their use, making them impractical in field settings or large insectaries where dozens of experimental crosses must be fed daily. Here we describe 3D-Feedy, an open-hardware, inexpensive membrane feeder that delivers electrically heated membrane feeding using simple, readily available parts. Powered via USB-C, including smartphone powerbanks, it is portable and straightforward to assemble with only basic tools and skills. In direct comparison with the widely used Hemotek feeder in Anopheles gambiae and Aedes albopictus, the 3D-Feedy achieved equal or higher feeding rates and significantly greater egg production while maintaining comparable larval hatching rates. These results validate 3D-Feedy as a practical and accessible tool for mosquito rearing and experimental studies. All design files and instructions are provided as open hardware resources.

zoology↗

bioRxiv2025

Developing Y chromosome sex ratio distorters in the model insect Drosophila melanogaster

CRISPR-Cas9 sex ratio distortion (SRD) systems can suppress insect populations by biasing progeny toward males, but realizing such systems requires reliable Cas9 expression from insect Y chromosomes. Here, we tested whether the spermatocyte-specific {beta}Tub85D promoter can drive functional Cas9 expression when inserted on the Drosophila melanogaster Y chromosome. Using CRISPR-mediated homology-directed repair, we generated a Y-linked {beta}Tub85D-Cas9-T2A-eGFP construct and compared its activity with an autosomal counterpart. Whereas autosomal {beta}Tub85D-Cas9 induced strong male-biased sex ratios when paired with an X-poisoning gRNA, the Y-linked construct failed to distort sex ratios and exhibited approximately 2,000-fold reduction in Cas9 transcript abundance. Nonetheless, weak but detectable GFP fluorescence and Cas9 transcripts confirmed partial Y-linked promoter activity. These findings provide the first direct experimental evidence of meiotic sex chromosome inactivation (MSCI) acting on the Drosophila Y chromosome, revealing that meiotic promoters can remain weakly active despite strong repression. This work defines transcriptional limits of the Drosophila Y chromosome and informs the design of next-generation Y-linked gene drives for sustainable insect control.

genetics↗

bioRxiv2025

An OpIE2-DsRed marker disrupts female blood-feeding and shortens lifespan in the malaria vector Anopheles gambiae.

Anopheles gambiae is one of the principal vectors of human malaria. Over the past two decades, transgenic mosquito strains have been essential tools for studying mosquito biology and developing genetic control strategies such as gene drives. Mosquito transformants are typically identified using fluorescent markers, which are assumed to be phenotypically neutral. While generating CRISPR-based gene drive strains carrying an OpIE2-DsRed marker we unexpectedly found that transgenic females were unable to blood-feed and were consequently sterile, whereas males initially appeared normal and fertile. Given the potential utility of dominant, female-specific sterility for mosquito control, we established additional strains controlling for transgene content and integration site, confirming that the OpIE2-DsRed cassette caused the defect. Behavioral assays showed that females exhibited normal attraction to a membrane feeder but failed to initiate blood-feeding, performing repeated cycles of probing and proboscis grooming in rapid succession before ultimately leaving the feeder unfed. Microscopy showed that both sexes possessed a distally curved proboscis, providing a morphological explanation for the blood-feeding defect of females and the reduced male lifespan. A second promoter variant (OpIE2b), differing in flanking sequences at the IE-2 junction, drove strong marker expression without impairing blood-feeding or longevity. These findings demonstrate that minor differences in promoter architecture can produce major, unexpected phenotypic effects. OpIE2b provides a robust, phenotypically neutral marker for An. gambiae research, while OpIE2a highlights the need for rigorous validation of transgenic components intended for research and applied releases.

genetics↗

bioRxiv2025

Comparative chemosensory mechanisms underlying larval foraging and competitive advantage in Aedes albopictus and Aedes aegypti

The invasive Asian tiger mosquito, Aedes albopictus (Skuse), and yellow fever mosquito, Aedes aegypti (L.) are known to compete for resources during the larval stage, often resulting in the ecological displacement of Ae. aegypti by Ae. albopictus. The chemosensory system plays a pivotal role in larval foraging behavior and may contribute to the competitive advantage. Here, we employed comparative transcriptomics and functional characterization of odorant receptors (ORs) to investigate species-specific differences in larval olfaction. Notably, we uncovered functional variation within the conserved olfactory indole receptor clade, indicating distinct ecological adaptations across species and life stages. We also developed a novel approach to functionally characterize the larval sensory cone and mapped its receptor neuron projections to two key brain regions: the antennal lobe and the subesophageal ganglion. This study provides new insights into the molecular and neural basis of chemosensory-driven behavior in mosquito larvae and highlights the potential role of olfaction in shaping interspecies competition and ecological success.

neuroscience↗

bioRxiv2025

CRISPR/Cas9-mediated mutagenesis of the white-eye gene in the tephritid pest Bactrocera zonata

Bactrocera zonata is a highly invasive agricultural pest that causes extensive damage to fruit crops. The Sterile Insect Technique (SIT), a species-specific and environmentally friendly pest control method, depends on the availability of Genetic Sexing Strains (GSSs) to enable efficient mass production of males for sterile release. However, no GSS currently exists for B. zonata limiting SIT applications targeting this important invasive pest. Here, we report two key advancements toward GSS development in this species. First, we present a high-quality, chromosome-level genome assembly from male B. zonata, identifying two scaffolds derived from the Y chromosome, which represent potential targets for future male-specific genetic engineering. Second, we demonstrate the feasibility of CRISPR/Cas9 genome editing in B. zonata by generating stable, homozygous white-eye mutants through targeted disruption of the conserved white-eye gene. This visible, recessive phenotype serves as a proof-of-concept for developing selectable markers in this species. Together, these results provide foundational genomic and genetic tools to support the development of GSSs in B. zonata, advancing the potential for sustainable, genetics-based pest control strategies.

bioengineering↗

bioRxiv2025

Mosquito Sex Separation using Complementation of Selectable Traits and Engineered Neo-Sex Chromosomes

Effective and scalable sex separation remains a critical challenge for mosquito genetic control strategies. Genetic sexing strains (GSS) address this by genetically linking maleness with selectable traits, enabling efficient removal of females before release. Here, we describe a robust platform for the development of GSSs in the invasive Aedes albopictus mosquito by integrating a CRISPR-engineered selectable phenotype with sex conversion via nix, the male-determining factor. As a proof-of-concept, we disrupt the yellow gene to generate a vivid pigmentation marker, then rescue its function in males using nix-containing transgenes, creating a stable strain where all females are yellow and all engineered males are dark. The resulting GSS males are fertile, robust, and despite lacking the ancestral M locus, exhibit gene expression profiles closely resembling wild-type males. We benchmark sex separation based on pigmentation and discover that yellow mutant females exhibit slower larval development, enhancing protandry-based sorting. The GSS strain is compatible with existing size-based sex sorting systems, allowing for improved separation accuracy through the integration of natural and engineered sexually dimorphic traits. Additionally, we find that GSS females lay desiccation-sensitive eggs, reducing the risk of accidental female releases. Our approach is the first to engineer a sex-linked selectable trait by precisely targeting an endogenous gene and restoring its function in males, establishing a versatile platform for GSS development in Aedes mosquitoes.

synthetic biology↗

bioRxiv2023

Revisiting Y-chromosome detection methods: R-CQ and KAMY efficiently identify Y chromosome sequences in Tephritidae insect pests

The detection and characterization of sex chromosome sequences is particularly important for major pest families, like the Tephritidae, whereas alternative pest management approaches, mainly involving male-only release programs, rely on the ability to target and manipulate sex-specific genomic regions, particularly those of the Y chromosome. However, resolving and detecting X and Y chromosome sequences at the chromosome level requires careful consideration of algorithmic outputs, especially in species where extensive sex chromosome markers are not available. Here, we present R-CQ and KAMY, two computational methods developed for the detection of sex chromosome-linked sequences. We evaluate their performance on newly generated chromosome-level assemblies of four important Tephritid pest species: Ceratitis capitata, Bactrocera dorsalis, Bactrocera zonata and Anastrepha ludens. By combining algorithmic predictions with a manual curation process, we assess the strengths and limitations of each method and provide a robust dataset of curated X- and Y-linked sequences. Overall, our results establish a framework for studying poorly characterized sex chromosome lineages and identifying sex-specific genomic regions, supporting the broader development of sex chromosome-based pest managements systems.

genomics↗

bioRxiv2023

Targeting mosquito X-chromosomes reveals complex transmission dynamics of sex ratio distorting gene drives

Engineered sex ratio distorters (SRDs) have been proposed as a powerful component of genetic control strategies designed to suppress harmful insect pests. Two types of CRISPR-based SRD mechanisms have been proposed: X-shredding eliminates X-bearing sperm, while X-poisoning eliminates daughters inheriting disrupted X-chromosomes. These differences can have a profound impact on the population dynamics of SRDs when linked to the Y-chromosome: an X-shredder is invasive constituting a classical meiotic Y-drive, whereas X-poisoning is self-limiting, unable to invade but also insulated from selection. Here, we established the first X-poisoning strains in the malaria vector Anopheles gambiae targeting three X-linked genes during spermatogenesis resulting in male bias. Surprisingly, we found that sex distortion was primarily driven by a loss of X-bearing sperm with limited evidence for postzygotic lethality of daughters. By leveraging a Drosophila melanogaster model, we show unambiguously that engineered SRD traits can operate differently in these two insects. Unlike X-shredding, X-poisoning could theoretically operate at early stages of spermatogenesis. We therefore explored premeiotic Cas9 expression to target the mosquito X-chromosome. We find that, by pre-empting the onset of meiotic sex chromosome inactivation, this approach may enable the development of Y-linked SRDs if mutagenesis of spermatogenesis-essential genes is functionally balanced.

synthetic biology↗

bioRxiv2023

Gene drive and genetic sex conversion in the global agricultural pest Ceratitis capitata

Homing-based gene drives are novel interventions promising the area-wide, species-specific genetic control of harmful insect populations. Here we characterise a first set of gene drives in a tephritid agricultural pest species, the Mediterranean fruit fly Ceratitis capitata (medfly). Our results show that the medfly is highly amenable to homing-based gene drive strategies. By targeting the medfly transformer gene, we also demonstrate two different mechanisms by which CRISPR-Cas9 gene drive can be coupled to sex conversion, whereby genetic females are transformed into fertile and harmless XX males. Given this unique malleability of sex determination, we modelled gene drive interventions that couple sex conversion and female sterility and found that such approaches could be effective and tolerant of resistant allele selection in the target population. Our results open the door for developing gene drive strains for the population suppression of the medfly and related tephritid pests by co-targeting female reproduction and shifting the reproductive sex ratio towards males. They demonstrate the untapped potential for gene drives to tackle agricultural pests in an environmentally friendly and economical way.

synthetic biology↗

bioRxiv2023

The evolution of borneol repellency in culicine mosquitoes

The use of essential oils derived from the camphor tree to repel mosquitoes is an ancient practice that originated in Southeast Asia and gradually spread to China and across Europe via the Maritime Silk Road. The olfactory mechanisms by which these oils elicit avoidance behavior are unclear. Here we show that plant bicyclic monoterpenoids and borneol specifically activate a neural pathway that originates in the orphan olfactory receptor neuron of the capitate peg sensillum in the maxillary palp, and projects to the mediodorsal glomerulus 3 in the antennal lobe. This neuron co-locates with two olfactory receptor neurons tuned to carbon dioxide and octenol that mediate human-host detection. We also confirm that borneol elicits repellency against human-seeking female mosquitoes. Understanding the functional role of the mosquito maxillary palp is essential to investigating olfactory signal integration and host-selection behavior.

evolutionary biology↗

bioRxiv2020

Engineered sex distortion in the global agricultural pest Ceratitis capitata

Genetic sex ratio distorters have potential for the area-wide control of harmful insect populations. Endonucleases targeting the X-chromosome and whose activity is restricted to male gametogenesis have recently been pioneered as a means to engineer such traits. Here we enabled endogenous CRISPR/Cas9 and CRISPR/Cas12a activity during spermatogenesis of the Mediterranean fruit fly Ceratitis capitata, a worldwide agricultural pest of extensive economic significance. In the absence of a chromosome-level assembly, we analysed long and short-read genome sequencing data from males and females to identify two clusters of abundant and X-chromosome specific sequence repeats. When targeted by gRNAs in conjunction with Cas9 they yielded a significant and consistent distortion of the sex ratio in independent transgenic strains and a combination of distorters induced a strong bias towards males (~80%). Our results demonstrate the design of sex distorters in a non-model organism and suggest that strains with characteristics suitable for field application could be developed for a range of medically or agriculturally relevant insect species.

synthetic biology↗

bioRxiv2020

Improved reference genome of the arboviral vector Aedes albopictus

The Asian tiger mosquito Aedes albopictus is globally expanding and has become the main vector for human arboviruses in Europe. Here we present AalbF2, a dramatically improved assembly of the Ae. albopictus genome that has revealed widespread viral insertions, novel microRNAs and piRNA clusters, the sex determining locus, new immunity genes, and has enabled genome-wide studies of geographically diverse Ae. albopictus populations and analyses of the developmental and stage-dependent network of expression data. Additionally, we built the first physical map for this species with 75% of the assembled genome anchored to the chromosomes. These up-to-date resources of the genome provide a foundation to improve understanding of the adaptation potential and the epidemiological relevance of this species and foster the development of innovative control measures. One Sentence SummaryLong-read and Hi-C-based de novo assembly of the arboviral vector Aedes albopictus genome fosters deeper understanding of its biological features.

genomics↗

bioRxiv2019

The potential for a released autosomal X-shredder becoming a driving-Y chromosome and invasively suppressing wild populations of malaria mosquitoes

Synthetic sex-ratio distorters based on X-chromosome shredding are predicted to be more efficient than sterile males for population suppression of malaria mosquitoes using genetic control. X-chromosome shredding operates through the targeted elimination of X-chromosome-bearing gametes during male spermatogenesis, resulting in males that have a high fraction of male offspring. Strains harboring autosomal constructs containing a modified endonuclease I-PpoI have now been developed in the malaria mosquito Anopheles gambiae, resulting in strong sex-ratio distortion towards males. Data are being gathered for these strains for submission of regulatory dossiers for contained use and subsequent field release in West Africa. Since autosomal X-shredders are transmitted in a Mendelian fashion and can be selected against their frequency in the population is expected to decline once releases are halted. However, any unintended transfer of the X-shredder to the Y-chromosome could theoretically change these dynamics: This could lead to 100% transmission of the newly Y-linked X-shredder to the predominant male-biased offspring and its insulation from negative selection in females, resulting in its potential spread in the population and ultimately to suppression. Here, we analyze plausible mechanisms whereby an autosomal X-shredder could become linked to the Y-chromosome after release and provide data regarding its potential for activity should it become linked to the Y-chromosome. Our results strongly suggest that Y-chromosome linkage through remobilization of the transposon used for the initial genetic transformation is unlikely, and that, in the unexpected event that the X-shredder becomes linked to the Y-chromosome, expression and activity of the X-shredder would likely be inhibited by meiotic sex chromosome inactivation. We conclude that a functioning X-shredding-based Y-drive resulting from a naturally induced transposition or translocation of the transgene onto the Y-chromosome is unlikely.

genetics↗

bioRxiv2019

A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters

Synthetic sex distorters have recently been developed in the malaria mosquito, relying on endonucleases that target the X-chromosome during spermatogenesis. Although inspired by naturally-occurring traits, it has remained unclear how they function and, given their potential for genetic control, how portable this strategy is across species. We established Drosophila models for two distinct mechanisms for CRISPR/Cas9 sex-ratio distortion - "X-shredding" and "X-meddling" - and dissected their target-site requirements and repair dynamics. X-shredding relies on sufficient meiotic activity of the endonuclease to overpower DNA repair and can operate on a single repeat cluster of non-essential sequences. X-meddling by contrast, i.e. targeting putative haplolethal X-linked genes, induced a bias towards males that is coupled to a loss in reproductive output, although a dominant-negative effect may drive the mechanism of female lethality. Our model system will guide the study and the application of sex distorters to medically or agriculturally important insect target species.

synthetic biology↗