Biology subjects

Alphey, L.

Publications and source records attributed to Alphey, L..

At least 19 recordsLinked to original sources

bioRxiv2026

Development and validation of an SDA-500 Anopheles stephensi cell line for molecular studies

BackgroundMalaria control is increasingly challenged by the urban-adapted vector Anopheles stephensi, yet molecular and cellular tools for this species remain scarce, restricting functional genomic studies and the development of genetic control strategies. To help address this gap, we established a new embryo-derived Anopheles stephensi cell line. ResultsWe generated and characterised a novel embryo-derived Anopheles stephensi (SDA-500) cell line capable of sustained growth in vitro. Species identity was confirmed by mitochondrial COI barcoding, and karyotypic analysis revealed a diploid chromosome complement with the presence of a Y chromosome, confirming that at least some cells are of male origin. Transfection conditions were optimized, with TransIT-PRO showing higher efficiency than Lipofectamine-based reagents. Using a dual-luciferase reporter assay, of several promoters tested the Anopheles gambiae polyubiquitin promoter exhibited the strongest and most consistent transcriptional activity in SDA-500 cells. ConclusionsThe SDA-500 cell line provides a stable and genetically validated in vitro platform that supports efficient transgene expression. This resource provides a useful system for functional genomics and molecular manipulation in Anopheles stephensi and is expected to facilitate studies of mosquito biology and contribute to the development of novel malaria control strategies.

cell biology↗

bioRxiv2026

Knock-in = knock-out: differential fitness effects of cardinal mutations in Anopheles stephensi

The kynurenine pathway metabolizes tryptophan into 3-hydroxykynurenine (3-HK), a precursor for ommochrome eye pigments synthesized via the cardinal (cd) gene in mosquitoes. While cd disruption was presumed neutral, we observed fitness costs in Anopheles stephensi knock-in but not knock-out cd mutants. Here we investigated this anomaly further by assessing survival, fecundity, and midgut integrity across multiple cd mutant lines. Heterozygous knock-in lines, expressing a fluorescent marker and guide RNA for CRISPR/Cas9, exhibited reduced survival post-blood feeding, larva-to-adult survival deficits, and midgut barrier dysfunction, whereas knock-outs showed no such costs. Oral supplementation with xanthurenic acid partially rescued knock-in mortality, implicating oxidative stress linked to 3-HK metabolism. Expression analyses suggest transgene insertion effects, rather than cd disruption, underlie these fitness costs. These findings highlight the importance of evaluating insertional effects in gene drive target selection and support cd as a viable target for genetic control strategies in An. stephensi.

genetics↗

bioRxiv2025

Defining the transmission parameters of lumpy skin disease virus by the three model insect vector species Aedes aegypti, Stomoxys calcitrans and Culicoides nubeculosus reveals important differences in their likely role in transmission

Lumpy skin disease virus (LSDV) is a poxvirus that can cause severe, systemic disease in cattle. By far the most important route of transmission of LSDV is mechanical transmission via haematophagous arthropod vectors. However we lack detailed information on this process including the likelihood of transmission by different vector species. This study used an experimental bovine model of LSDV transmission to quantify the transmission of LSDV from an infected donor to a naive recipient calf. Three species of Diptera representing different vector groups were included (Stomoxys calcitrans, Aedes aegypti and Culicoides nubeculosus, respectively a large biting fly, a mosquito and a midge), and the clinical, virological and immunological outcomes in the recipient calves studied. The ability of Ae. aegypti to mechanically transmit LSDV following feeding on an artificial membrane feeding system was also examined. Both Ae. aegypti and S. calcitrans were able to transmit LSDV, resulting in disease in recipient calves. Bites from virus-positive C. nubeculosus did not result in disease in recipient calves, though the presence of neutralising antibodies in these recipients indicated exposure to virus or virus components. Ae. aegypti successfully transmitted LSDV following feeding on LSDV-spiked blood through an artificial membrane feeding system, validating this laboratory model as a future replacement for donor cattle. Mathematical models of the data were generated and predicted S. calcitrans to be the most efficient vector of LSDV of the insects tested with a reproduction number (R0) of 5.8. Impact statementLumpy skin disease virus (LSDV) is a neglected, rapidly emerging pathogen of cattle that has spread into Europe and throughout the Middle East and Asia over the past ten years. Lack of understanding of the mechanism of transmission of LSDV has hampered efforts to control its rapid spread. This study compares the ability of three model species of Diptera (Stomoxys calcitrans, Aedes aegypti and Culicoides nubeculosus) to mechanically transmit the virus to cattle, generating high quality quantitative data to facilitate mathematical modelling of virus transmission. S. calcitrans was identified as a potential driver of LSDV transmission with a R0 of 5.8. This work provides new insights into the vector transmission of LSDV that can be used to design more targeted and effective disease control programmes.

microbiology↗

bioRxiv2025

Integrating multiplexing into confineable gene drives effectively overrides resistance in Anopheles stephensi

Anopheles stephensi is a major malaria vector mainly present in southern Asia and the Arabian Peninsula. Since 2012 it has invaded several countries of eastern Africa, stimulating urgent efforts to develop more efficient strategies for vector control such as CRISPR/Cas9-based homing gene drives. Target site resistance is a significant challenge to the deployment of these systems. When a double-stranded break is repaired by NHEJ, it can lead to mutations which destroy the target site, making that allele unrecognizable to the sgRNA and resistant to further cleavage. The use of multiple sgRNAs has the potential to solve this issue. We performed experimental crosses to assess the homing and cutting efficiency of two different multiplexing strategies targeting the cardinal locus, in the presence and absence of a resistance allele. We found pre-existing mutations at one sgRNA target site did not significantly reduce the homing efficiency for either strategy. Modelling indicates that while both strategies can overcome resistance allele formation, the fitness of the drive-carrying alleles is a critical factor in determining the overall performance and persistence of a split drive.

genetics↗

bioRxiv2025

Sequence mismatch between gene-drive and target-site flanking regions significantly impairs homing efficiency in Culex quinquefasciatus

CRISPR/Cas9-based homing gene-drives (homing-drives) hold enormous potential as control tools for mosquito disease-vectors. These genomically-encoded technologies spread themselves through target populations by creating double-stranded DNA breaks on homologous chromosomes, into which the homing-drives are copied ( homed). Homing is dependent on sequence homology between the genomic regions flanking the transgene insertion and the break site. Homing efficiency (i.e. copying rate) substantially impacts the power of these systems: less efficient homing-drives spread slower, have fewer applications and are more resistance-prone. Understanding what influences homing-drive efficiency is therefore vital to the successful use of these technologies. Here we report a novel mechanism by which a homing-drives efficiency can be significantly impaired by natural sequence variation within a population into which it is spreading. Using a kmo-targeting split homing-drive in the West Nile virus mosquito Culex quinquefasciatus, we found that target-site heterology (sequence mismatch between the genomic regions flanking the target cut-site and the homing-drive transgene) of less than 10% reduced homing efficiency by up to 54%. While substantial research effort has been dedicated to increasing homing-drive efficiency through optimisation of within-construct components, our results highlight that the real-world efficacy of these systems may in part depend on variation beyond these controllable factors.

synthetic biology↗

bioRxiv2023

Transgenic expression of cif genes from Wolbachia strain wAlbB recapitulates cytoplasmic incompatibility in Aedes aegypti.

The endosymbiotic bacteria Wolbachia can invade insect populations by modifying host reproduction through cytoplasmic incompatibility (CI), an effect that results in embryonic lethality when Wolbachia-carrying males mate with Wolbachia-free females. Here we describe a transgenic system for recreating CI in the major arbovirus vector Aedes aegypti using CI factor (cif) genes from wAlbB, a Wolbachia strain currently being deployed to reduce dengue transmission. CI-like sterility was induced when cifA and cifB were co-expressed in testes; this sterility could be rescued by maternal cifA expression, thereby reproducing the pattern of Wolbachia-induced CI. Expression of cifB alone was associated with extensive DNA damage and disrupted spermatogenesis. The strength of rescue by maternal cifA expression was dependent on the comparative levels of cifA/cifB expression in males. These findings are consistent with CifB acting as a toxin and CifA as an antitoxin, with CifA attenuating CifB toxicity in both the male germline and in developing embryos.

microbiology↗

bioRxiv2023

A synthetic biology approach to transgene expression

The ability to control gene expression is pivotal in genetic engineering and synthetic biology. However, in most non-model and pest insect species, empirical evidence for predictable modulation of gene expression levels is lacking. This knowledge gap is critical for genetic control systems, particularly in mosquitoes, where transgenic methods offer novel routes for pest control. Commonly, the choice of RNA polymerase II promoter (Pol II) is the primary method for controlling gene expression, but the options are limited. To address this, we developed a systematic approach to characterize modifications in translation initiation sequences (TIS) and 3 untranslated regions (UTR) of transgenes, enabling the creation of a toolbox for gene expression modulation in mosquitoes and potentially other insects. The approach demonstrated highly predictable gene expression changes across various cell lines and promoter sequences, representing a significant advancement in mosquito synthetic biology gene expression. tools. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/555539v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1db954forg.highwire.dtl.DTLVardef@1279543org.highwire.dtl.DTLVardef@1b86fa2org.highwire.dtl.DTLVardef@1a9176a_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

AePUb promoter length modulates gene expression in Aedes aegypti

Molecular tools for modulating transgene expression in Aedes aegypti are few. Here we demonstrate that adjustments to the AePUb promoter length can alter expression levels of two reporter proteins in Ae. aegypti cell culture and in mosquitoes. This provides a simple means for increasing or decreasing expression of a gene of interest and easy translation from cells to whole insects.

molecular biology↗

bioRxiv2023

Optimizing CRE and PhiC31 mediated recombination in Aedes aegypti

Genetic manipulation of Aedes aegypti is key to developing a deeper understanding of this insects biology, vector-virus interactions and makes future genetic control strategies possible. Despite some advances, this process remains laborious and requires highly skilled researchers and specialist equipment. Here we present two improved methods for genetic manipulation in this species. Use of transgenic lines which express Cre recombinase allowed, by simple crossing schemes, germline or somatic recombination of transgenes, which could be utilized for numerous genetic manipulations. PhiC31 integrase based methods for site-specific integration of genetic elements was also improved, by developing a plasmid which expresses PhiC31 when injected into early embryos, eliminating the need to use costly and unstable mRNA as is the current standard.

bioengineering↗

bioRxiv2023

CRISPR-based gene drives generate super-Mendelian inheritance in the disease vector Culex quinquefasciatus.

Culex mosquitoes pose a significant public health threat as vectors for a variety of diseases including West Nile virus and lymphatic filariasis, and transmit pathogens threatening livestock, companion animals, and endangered birds. Rampant insecticide resistance makes controlling these mosquitoes challenging and necessitates the development of new control strategies. Gene drive technologies have made significant progress in other mosquito species, although similar advances have been lagging in Culex. Here we test the first CRISPR-based homing gene drive for Culex quinquefasciatus, demonstrating the possibility of using this technology to control Culex mosquitoes. Our results show that the inheritance of two split-gene-drive transgenes, targeting different loci, are biased in the presence of a Cas9-expressing transgene although with modest efficiencies. Our findings extend the list of disease vectors where engineered homing gene drives have been demonstrated to include Culex alongside Anopheles and Aedes, and pave the way for future development of these technologies to control Culex mosquitoes.

synthetic biology↗

bioRxiv2023

A Zika virus-responsive sensor-effector system in Aedes aegypti

Zika virus (ZIKV) is a recently re-emerged flavivirus transmitted primarily through the bite of an infected mosquito, Aedes aegypti being the main vector. ZIKV infection is associated with a range of adverse effects; infection during pregnancy can lead to foetal abnormalities, including microcephaly. Lacking a licensed vaccine, or specific therapeutics, control of ZIKV transmission focuses on vector control. However, in most transmission settings, current methods are insufficient to successfully control ZIKV, or other similarly-transmitted arboviruses such as dengue and chikungunya viruses. This has stimulated interest in genetics-based methods, either to reduce the number of mosquitoes ("population suppression"), or to make mosquitoes less able to transmit ("population modification"). Here, we describe a method to selectively eliminate infected mosquitoes, using a virus sensor inserted into the mosquito genome and coupled to a quorum-counting lethal effector. In mosquitoes, ZIKV normally establishes persistent, lifelong infection; survival of these infected mosquitoes is crucial to transmission potential. Correspondingly, removal of infected mosquitoes can reduce vectorial capacity of a mosquito population, i.e. ability to transmit. Since relatively few mosquitoes become infected, typically <2%, engineered hypersensitivity to ZIKV would have only a modest population-level fitness cost, and lower still if transmission were successfully reduced by such means.

bioengineering↗

bioRxiv2022

Fake it to break it: mimicking superinfection exclusion disrupts alphavirus infection and transmission in the yellow fever mosquito Aedes aegypti

Multiple viruses cause a phenomenon termed superinfection exclusion whereby a currently infected cell is resistant to secondary infection by the same or a closely related virus. In alphaviruses, this process is thought to be mediated, at least in part, by the viral protease (nsP2) which is responsible for processing the non-structural polyproteins (P123 and P1234) into individual proteins (nsP1-nsP4), forming the viral replication complex. Taking a synthetic-biology approach, we mimicked this naturally occurring phenomenon by generating a superinfection exclusion-like state in Aedes aegypti mosquitoes, rendering them refractory to alphavirus infection. By artificially expressing Sindbis virus (SINV) and chikungunya virus (CHIKV) nsP2 in mosquito cells and transgenic mosquitoes, we demonstrated a reduction in both SINV and CHIKV viral replication rates in cells following viral infection as well as reduced infection prevalence, viral titres and transmission potential in mosquitoes.

microbiology↗

bioRxiv2022

A multiplexed, confinable CRISPR/Cas9 gene drive propagates in caged Aedes aegypti populations

Aedes aegypti, the yellow fever mosquito, is the main vector of several major pathogens including yellow fever, dengue, Zika and chikungunya viruses. Classical mosquito control strategies, mainly utilizing insecticides, have had success in controlling other mosquito vectors in recent years, but are much less useful against Ae. aegypti, and even these methods are threatened by rising insecticide resistance. This has stimulated interest in new mosquito control mechanisms, notably genetic systems such as gene drives. However, the development of CRISPR/Cas9 gene drive systems has faced challenges such as low inheritance biasing rate, the emergence of resistance alleles, and the possibility of spreading beyond the intended population. Here, we test the regulatory sequences from the Ae. aegypti benign gonial cell neoplasm (bgcn) homolog to express Cas9 in the germline to find an expression timing more conducive to homing. We also created a separate multiplexing (targeting multiple different sites within the target gene) sgRNA-expressing homing cassette inserted into the Ae. aegypti kynurenine 3-monooxygenase (kmo) gene to limit the consequences of resistance alleles. This creates a split gene drive such that one part does not drive, allowing control over geographic spread and temporal persistence. When combined, these two elements provide highly effective germline cutting at the kmo locus and act as a gene drive. Our target genetic element was driven through a cage trial population such that carrier frequency of the element increased from 50% to up to 89% of the population despite significant fitness costs to kmo insertions. Deep sequencing suggests that the multiplexing design could mitigate resistance allele formation in our gene drive system. Significance statementMosquito-borne diseases affect millions of people worldwide, with the yellow fever mosquito (Aedes aegypti) being the principal vector of many viral diseases. Effective measures for controlling this mosquito are sorely needed. Gene drive systems have arisen as a potential tool for mosquito control due to their ability of biasing inheritance of a trait into a target population. Here, we assess a split gene drive, based on CRISPR/Cas9 endonuclease technology driving a target element into the mosquito population. Evaluated over successive generations in a replicated cage trial, the drive successfully biased its inheritance, increasing in frequency from 50% to up to 89%. Our results are encouraging for the potential use of this type of contained gene drive system for mosquito control in endemic areas.

synthetic biology↗

bioRxiv2022

Small silencing RNAs expressed from W-linked retrocopies of Masculinizer target the male-determining gene PxyMasc during female sex determination in the Diamondback moth Plutella xylostella

The Lepidoptera are an insect order of cultural, economic and environmental importance, representing c. 10% of all described living species. Yet, for all but one of these species (silkmoth, Bombyx mori) the molecular genetics of how sexual fate is determined remains unknown. We investigated this in the diamondback moth (DBM - Plutella xylostella), a globally important, highly invasive and economically damaging pest of cruciferous crops. Our previous work uncovered a regulator of male sex determination in DBM - PxyMasc, a homologue of B. mori Masculinizer - which although initially expressed in embryos of both sexes, is then reduced in female embryos, leading to female-specific splicing of doublesex. Here, through sequencing small RNA libraries generated from early embryos and sexed larval pools, we identified a variety of small silencing RNAs (predominantly piRNAs) complementary to PxyMasc, whose temporal expression correlated with the reduction in PxyMasc transcript observed previously in females. Analysis of these small RNAs showed that they are expressed from tandemly-arranged, multi-copy arrays found exclusively on the W (female-specific) chromosome, which we term Pxyfem. Analysis of the Pxyfem sequences showed that they are partial cDNAs of PxyMasc mRNA transcripts, likely integrated into transposable element graveyards by the non-canonical action of retrotransposons (retrocopies), and that their apparent similarity to B. mori feminizer more probably represents convergent evolution. Our study helps elucidate the sex determination cascade in this globally important pest and highlights the shortcuts which retrotransposition events can facilitate in the evolution of complex molecular cascades, including sex determination. Significance statementUncovering the mechanisms which species have evolved to determine sex is of fundamental interest and provides avenues for pest management through genetic manipulation of these pathways. In insects, much of what is known regarding sex determination is concentrated within the Diptera and Hymenoptera, despite other orders (e.g. Lepidoptera) being of great ecological and economic importance. Here, using small RNA sequencing of embryonic and early larval samples, we uncover an RNAi-based sex determination system which silences the male determining gene PxyMasc in the Diamondback moth (Plutella xylostella) - a global pest of cruciferous crops. We track production of these silencing RNAs back to the W-chromosome where they are expressed from partial cDNA copies of PxyMasc. Our analysis suggests these are PxyMasc retrocopies, integrated via the non-canonical action of LTR retrotransposons and that similarities between this system and the feminizer system in Bombyx mori likely represent convergent evolution.

developmental biology↗

bioRxiv2021

Towards CRISPR/Cas9-based gene drive in the diamondback moth Plutella xylostella

Promising to provide powerful genetic control tools, gene drives have been constructed in multiple dipterans, yeast and mice, for the purposes of population elimination or modification. However, it remains unclear whether these techniques can be applied to lepidopterans. Here, we used endogenous regulatory elements to drive Cas9 and sgRNA expression in the diamondback moth, (Plutella xylostella), and test the first split-drive system in a lepidopteran. The diamondback moth is an economically important global agriculture pest of cruciferous crops and has developed severe resistance to various insecticides, making it a prime candidate for such novel control strategy development. A very high level of somatic editing was observed in Cas9/sgRNA transheterozygotes, although no significant homing was revealed in the subsequent generation. Although heritable, Cas9-medated germline cleavage, as well as maternal and paternal Cas9 deposition was observed, rates were far lower than for somatic cleavage events, indicating robust somatic but limited germline activity of Cas9/sgRNA under the control of selected regulatory elements. Our results provide valuable experience, paving the way for future construction of gene drive-based genetic control strategies in DBM or other lepidopterans.

genetics↗

bioRxiv2021

CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say

Culex quinquefasciatus Say is a brown, medium sized mosquito distributed widely in both tropical and subtropical regions of the world. It is a night-active, opportunistic blood-feeder and is responsible for vectoring many animal and human diseases, including West Nile Virus and avian malaria. Current vector control methods (e.g. physical / chemical) are increasingly ineffective; use of insecticides also imposes some hazards to both human and ecosystem health. Recent advances in genome editing have allowed the development of genetic methods of insect control, which is species-specific and, theoretically, highly effective. CRISPR/Cas9 is a bacteria-derived programmable gene editing tool that has been shown to be functional in a range of species. We demonstrate here, the first successful germline gene knock-in by homology dependent repair in C. quinquefasciatus. Using CRISPR/Cas9, we integrated exogenous sequence comprising a sgRNA expression cassette and marker gene encoding a fluorescent protein fluorophore (Hr5/IE1-DsRed, Cq7SK-sgRNA) into the kynurenine 3-monooxygenase (kmo) gene. We achieved a minimum transformation rate of 2.8% similar to rates achieved in other mosquito species. Precise knock-in at the intended locus was confirmed by sequencing. Insertion homozygotes displayed a white eye phenotype in early-mid stage larvae and a recessive lethal phenotype by pupation. This work shows an alternative and efficient method for genetic engineering of C. quinquefasciatus, providing a new tool for researchers interested in developing genetic control tools for this vector.

bioengineering↗

bioRxiv2020

A CRISPR endonuclease gene drive reveals two distinct mechanisms of inheritance bias

RNA guided CRISPR gene drives have shown the capability of biasing transgene inheritance in multiple species. Among these, homing endonuclease drives are the most developed. In this study, we report the functioning of sds3, bgcn, and nup50 expressed Cas9 in an Aedes aegypti homing split drive system targeting the white gene. We report their inheritance biasing capability, propensity for maternal deposition, and zygotic/somatic expression. Additionally, by making use of the tight linkage of white to the sex-determining locus, we were able to elucidate mechanisms of inheritance bias. We find inheritance bias through homing in double heterozygous males, but find that a previous report of the same drive occurred through meiotic drive. We propose that other previously reported homing design gene drives may in fact bias their inheritance through other mechanisms with important implications for gene drive design.

genetics↗

bioRxiv2020

Engineered expression of the invertebrate-specific scorpion toxin AaHIT reduces adult longevity and female fecundity in the diamondback moth Plutella xylostella

BACKGROUNDPrevious Genetic Pest Management (GPM) systems in diamondback moth (DBM) have relied on expressing lethal proteins ( effectors) that are cell-autonomous i.e. do not leave the cell they are expressed in. To increase the flexibility of future GPM systems in DBM, we aimed to assess the use of a non cell-autonomous, invertebrate-specific, neurotoxic effector - the scorpion toxin AaHIT. This AaHIT effector was designed to be secreted by expressing cells, potentially leading to effects on distant cells, specifically neuromuscular junctions. RESULTSExpression of AaHIT caused a shaking/quivering phenotype which could be repressed by provision of an antidote (tetracycline); a phenotype consistent with the AaHIT mode-of-action. This effect was more pronounced when AaHIT expression was driven by the Hr5/ie1 promoter (82.44% of males, 65.14% of females) rather than Op/ie2 (57.35% of males, 48.39% of females). Contrary to expectations, the shaking phenotype and observed fitness costs were limited to adults where they caused severe reductions in mean longevity (-81%) and median female fecundity (-93%). qPCR of AaHIT expression patterns and analysis of piggyBac-mediated transgene insertion sites suggest that restriction of observed effects to the adult stages may be due to influence of local genomic environment on the tetO-AaHIT transgene. CONCLUSIONWe have demonstrated the feasibility of using non cell-autonomous effectors within a GPM context for the first time in the Lepidoptera, one of the most economically damaging orders of insects. These findings provide a framework for extending this system to other pest Lepidoptera and to other secreted effectors.

genetics↗