Biology subjects

Maselko, M.

Publications and source records attributed to Maselko, M..

14 recordsLinked to original sources

bioRxiv2025

The LAST mile: Evaluating genetic biocontrol as a supplemental tool for eradicating invasive rodents on islands

Invasive rodents cause severe ecosystem degradation on islands and can be challenging to eradicate. Current best-practices rely heavily on the application of toxic oral baits and have led to successful eradications and remarkable recoveries of native flora and fauna. Yet this single method is not universally applicable. Reliance on a single method limits further eradication success in situations where toxicants cannot be used or remnant populations persist after application. Genetic biocontrol offers a suite of new solutions to potentially improve outcomes in the critical "last mile" of eradication. These approaches involve the release of genetically modified individuals of the target species to reduce population fitness over time. These include self-limiting approaches which require multiple releases and self-sustaining mechanisms (i.e., select gene-drive systems) which could theoretically collapse populations after a single release. While gene drive systems have received significant attention, their development in vertebrates remains technically challenging, and their ecological and regulatory implications are still in active debate. In contrast, some non-drive genetic biocontrol approaches, such as Y-linked editors, fsRIDL, and Gravid Lethal, offer self-limiting alternatives that may be more immediately deployable. These approaches could be used to supplement toxicant-based methods and may also have reduced environmental risks and regulatory barriers. To evaluate the potential of these tools, we developed an individual-based model simulating the eradication of house mice (Mus musculus) on a 125ha island with an initial population of 11,000 individuals. We tested various combinations of genetic biocontrol release and effort strategies to understand tradeoffs between required effort and uncertainty for achieving successful eradication. Under certain effort strategies, we found that the Gravid Lethal approach performed the best, achieving eradication in less than 2.3 years with intensive release effort and monitoring. Our findings suggest that integrating non-drive genetic biocontrol into adaptive management frameworks could enhance the effectiveness and feasibility of rodent eradication programs. These tools are not replacements for toxicants but may serve as critical supplements--particularly in the "last mile" of eradication. Further stakeholder engagement is needed to assess the ecological, ethical, and logistical dimensions of deploying these technologies in real-world settings.

ecology↗

bioRxiv2025

Heterologous expression of microbial nitroreductases for TNT degradation in transgenic animals

TNT (2,4,6-trinitrotoluene) from unexploded ordinances is a common environmental pollutant near munitions factories, military training sites, and areas of armed conflict. As physical and chemical approaches for TNT remediation are costly and difficult to scale, in situ bioremediation is an attractive alternative. TNT is a phytoaccumulative pollutant. Herbivores engineered to detoxify TNT are a potentially cost-effective platform to bioremediate large areas of contaminated sites while grazing or browsing. As a first step towards engineering herbivores with metabolic capabilities for TNT degradation, we engineered the animal genetic model, Drosophila melanogaster, to screen a library of microbial nitroreductases that can catalyse the initial degradation steps required for TNT degradation. We find strong, cofactor-dependent activity in fly lysates engineered to express NsfA from Escherichia coli, and NsfI from Enterobacter cloacae.

synthetic biology↗

bioRxiv2025

Efficient CRISPR-Cas9-mediated genome editing of the cane toad (Rhinella marina)

Invasive species inflict major ecological, economic, social, and cultural harm worldwide, highlighting the urgent need for innovative and effective control strategies. Genome editing offers exciting possibilities for creating highly targeted control methods for invasive species. Here, we demonstrate CRISPR-Cas9 genome editing in the cane toad (Rhinella marina), one of Australias most notorious invasive species, by targeting the tyrosinase gene to produce albino phenotypes that provide clear visual markers for assessing editing efficiency. Microinjection of Cas9 protein and guide RNAs into one-cell zygotes resulted in 87.6% of mosaic larvae displaying nearly complete albinism, with 2.3% exhibiting complete albinism. For completely albino individuals, genomic analysis confirmed predominantly frameshift mutations or large deletions at the target site, with no wild-type alleles detected. Germline transmission rates reflected the extent of albinism in the mosaic adult, where we achieved maternal germline transmission rates of almost 100%. This technology, representing the first application of CRISPR-Cas9 in the Bufonidae family, opens possibilities for exploring both basic research questions and strategies for population control.

synthetic biology↗

bioRxiv2024

Optimization of rearing Transeius montdorensis under laboratory conditions

The global application of Transeius montdorensis (Acari: Phytoseiidae) as a biological control agent across various protected crops has proven effective against a range of insect pests like thrips and whiteflies, as well as pest mites like broad mites and russet mites. Optimization of rearing T. montdorensis under laboratory conditions is crucial for further studies of this species to improve their application in Integrated Pest Management (IPM) programs. Here, we evaluated the development and reproduction of T. montdorensis when fed on four different diets, including cattail pollen (Typha latifolia), living dried fruit mites (Carpoglyphus lactis), frozen C. lactis eggs, and a mixed diet of frozen C. lactis eggs and T. latifolia pollen. Females consuming the mixed diet exhibited superior total fecundity and daily oviposition rate, along with the highest intrinsic rate of increase (rm) and net productive rate (R0) among all diets tested. The immature period was significantly longer for mites on a diet of living C. lactis compared to those on other diets. Importantly, utilizing frozen C. lactis eggs and T. latifolia pollen mitigates the risk of infestation and contamination from the living dried fruit mites, which is important for laboratory and field settings when releasing the predator colonies. Our findings not only present an optimized rearing method for predatory mites under laboratory conditions but also suggest potential broader applications for enhancing the effectiveness and sustainability of biological control strategies across various agroecosystems and reducing dependency on chemical pesticides.

zoology↗

bioRxiv2024

Biomanufacturing of a functional microbial phytase in an insect host.

BackgroundInsects, such as Black Soldier Flies (Hermetia illucens), are increasingly used as sustainable animal feed ingredients that can be reared on plentiful organic substrates such as agricultural residues and pre-consumer food waste. Genetically engineering insects to heterologously express feed additive enzymes has the potential to generate more value from organic waste, while improving livestock health and productivity. Phytases are widely used feed additive enzymes that hydrolyse the phosphate groups from the myo-inositol backbone of phytic acid, a phosphate rich antinutrient compound that monogastric animals cannot efficiently digest. Dietary phytase supplementation improves absorption of phosphorous, proteins, and cationic nutrients, while mitigating the negative environmental effects of phytic acid rich excreta. ResultsWe evaluated the potential of using insects to biomanufacture microbial feed additive enzymes by engineering the model insect, Drosophila melanogaster, to express phytases. One histidine acid phytase, three beta propellor phytases, three purple acid phosphatases, and one PTP-like phytase were selected for screening in D. melanogaster. Transgenic flies expressing the AppA histidine acid phytase from E. coli had 27.82 FTU/g of phytase activity, which exceeds the 0.5-1.0 FTU/g required in animal feed. Maximum activity from AppA phytase expressed by D. melanogaster was observed at pH 5 and 55 {degrees}C, however, more than 50% of phytase activity was present at 25 {degrees}C and pH 2. Here we demonstrate that insects may be suitable hosts for the heterologous expression of a microbial phytase enzyme with applications for improving animal feed nutrition and organic waste valorisation.

synthetic biology↗

bioRxiv2024

Allele Sails: launching traits and fates into wild populations with DNA sequence modifiers

Population-scale genome editing can be used to alter the composition or fate of wild populations. One approach to achieving these aims utilizes a synthetic gene drive element--a multi-gene cassette--to bring about an increase in the frequency of an existing allele. However, the use of gene drives is complicated by the multiple scientific, regulatory, and social issues associated with transgene persistence and gene flow. Alternatives in which transgenes are not driven could potentially avoid some of these issues. Here we propose an approach to population scale gene editing using a system we refer to as an Allele Sail. An Allele Sail consists of a genome editor (the Wind) that introduces DNA sequence edits (the Sail) at one or more sites, resulting in progeny that are viable and fertile. The editor, such as a sequence-specific nuclease, or a prime- or base-editor, is inherited in a Mendelian fashion. Meanwhile, the edits it creates experience an arithmetic, Super-Mendelian increase in frequency. We explore this system using agent-based modeling, and identify contexts in which a single, low frequency release of an editor brings edits to a very high frequency. We also identify conditions in which manipulation of sex determination can be used to bring about population suppression. Current regulatory frameworks often distinguish between transgenics as genetically modified organisms (GMOs), and their edited non-transgenic progeny as non-GMO. In this context an Allele Sail provides a path to alter traits and fates of wild populations in ways that may be considered more acceptable.

synthetic biology↗

bioRxiv2024

Rapid generation and screening of transgenic black soldier fly (Hermetia illucens)

BackgroundThe black soldier fly (BSF), Hermetia illucens is a widely used, and mass-produced insect that fulfils an important role in both the management of organic waste and as a component of animal feed formulations. They also have significant potential as a platform for converting organic waste into high-value proteins, and lipids for the production of biofuels. Applying synthetic biology to BSF provides even more potential for improvement through the generation of transgenic BSF to enhance animal feed, produce and fine tune high-value industrial biomolecules, and to expand their waste conversion capabilities. ResultsTo enable the rapid generation and screening of transgenic BSF, we utilised microinjections of piggyBac mRNA with donor plasmids. We have found preliminary screening of G0 BSF to identify mosaics for outcrossing can be completed less than 2 weeks after microinjection. Stable transgenic lines were reliably generated with effective transformation rates of 30-33%, and transmission of the transgene could be confirmed 3 days after outcrossing the G0 adults. We also present a protocol for identifying the location of integrated transgenes. ConclusionsThe methods presented here expedite the screening process for BSF transgenesis and further expand the toolkit for BSF synthetic biology.

synthetic biology↗

bioRxiv2024

Recombinant venom proteins in insect seminal fluid reduces female lifespan

The emergence of insecticide resistance has increased the need for alternative pest management tools1,2 Numerous genetic biocontrol approaches, which involve the release of genetically modified organisms to control pest populations, are in various stages of development to provide highly targeted pest control3-7. However, all current mating-based genetic biocontrol technologies function by releasing engineered males which skew sex-ratios or reduce offspring viability in subsequent generations. This allows mated females continue to cause harm (e.g. transmit disease). Here, we demonstrate the first example of intragenerational genetic biocontrol, wherein mating with engineered males reduces female lifespan. The toxic male technique (TMT) involves the heterologous expression of insecticidal proteins within the male reproductive tract that are transferred to females via mating. We demonstrate TMT in Drosophila melanogaster males, which reduce the median lifespan of mated females by 37 - 59% compared to controls mated to wild type males. Agent-based models of Aedes aegypti predict that compared to existing genetic biocontrol technologies, even modest levels of mated female mortality could allow TMT to suppress a female population substantially faster, which is likely to result in reduced disease burdens. TMT presents a novel approach for combatting outbreaks of disease vectors and agricultural pests. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/575309v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@9930eforg.highwire.dtl.DTLVardef@15bbc0borg.highwire.dtl.DTLVardef@1967af9org.highwire.dtl.DTLVardef@63a589_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Methylmercury demethylation and volatilization by animals expressing microbial enzymes.

Methylmercury is a highly toxic pollutant that accumulates in food webs where it is inaccessible to current remediation technologies. We demonstrate that animals can be engineered to express the microbial enzymes, organomercurial lyase (MerB) and mercuric reductase (MerA), to bioremediate methylmercury. MerA and MerB from Escherichia coli were functional in invertebrate (Drosophila melanogaster) and vertebrate (Danio rerio) model systems and converted methylmercury into volatile Hg0. The engineered animals tolerated higher exposures to methylmercury and accumulated less than half as much mercury relative to their wild-type counterparts. The outcomes of this research could be applied to reduce mercury contamination in farmed and recreationally caught fish, for species conservation, and to restore value to organic wastes contaminated with mercury.

synthetic biology↗

bioRxiv2021

Genetically engineered insects with sex-selection and genetic incompatibility enable population suppression

Engineered Genetic Incompatibility (EGI) is a method to create species-like barriers to sexual reproduction. It has applications in pest control that mimic Sterile Insect Technique when only EGI males are released. This can be facilitated by introducing conditional female-lethality to EGI strains to generate a sex-sorting incompatible male system (SSIMS). Here we demonstrate a proof of concept by combining tetracycline-controlled female lethality constructs with a pyramus-targeting EGI line in the model insect Drosophila melanogaster. We show that both functions (incompatibility and sex-sorting) are robustly maintained in the SSIMS line and that this approach is effective for population suppression in cage experiments. Further we show that SSIMS males remain competitive with wild-type males for reproduction with wild-type females, including at the level of sperm competition.

bioengineering↗

bioRxiv2021

Bioremediation of industrial pollutants by insects expressing a fungal laccase

Inadequate management of household and industrial wastes pose major challenges to human and environmental health. Advances in synthetic biology may help address these challenges by engineering biological systems to perform new functions such as biomanufacturing of high-value compounds from low-value waste streams and bioremediation of industrial pollutants. The current emphasis on microbial systems for biomanufacturing, which often require highly pre-processed inputs and sophisticated infrastructure, is not feasible for many waste streams. Concerns about transgene biocontainment have limited the release of engineered microbes or plants for bioremediation. Engineering animals may provide opportunities for utilizing various waste streams that are not suitable for microbial biomanufacturing while effective transgene biocontainment options should enable in situ bioremediation. Here, we engineer the model insect Drosophila melanogaster to express a functional laccase from the fungus Trametes trogii. Laccase expressing flies reduced concentrations of the endocrine disruptor bisphenol A by more than 50% when present in their growth media. A lyophilized powder made from engineered adult flies retained substantial enzymatic activity, degrading more than 90% of bisphenol A and the textile dye indigo carmine in aqueous solutions. Our results demonstrate that transgenic animals may be used to bioremediate environmental contaminants in vivo and serve as novel production platforms for industrial enzymes. These results support further development of insects, and possibly other animals, as bioproduction platforms and their potential use in bioremediation.

synthetic biology↗

bioRxiv2020

Engineering multiple species-like genetic incompatibilities in insects

Speciation constrains the flow of genetic information between populations of sexually reproducing organisms. Gaining control over mechanisms of speciation would enable new strategies to manage wild populations of disease vectors, agricultural pests, and invasive species. Additionally, such control would provide safe biocontainment of transgenes and gene drives. Natural speciation can be driven by pre-zygotic barriers that prevent fertilization or by post-zygotic genetic incompatibilities that render the hybrid progeny inviable or sterile. Here we demonstrate a general approach to create engineered genetic incompatibilities (EGIs) in the model insect Drosophila melanogaster. Our system couples a dominant lethal transgene with a recessive resistance allele. EGI strains that are homozygous for both elements are fertile and fecund when they mate with similarly engineered strains, but incompatible with wild-type strains that lack resistant alleles. We show that EGI genotypes can be tuned to cause hybrid lethality at different developmental life-stages. Further, we demonstrate that multiple orthogonal EGI strains of D. melanogaster can be engineered to be mutually incompatible with wild-type and with each other. Our approach to create EGI organisms is simple, robust, and functional in multiple sexually reproducing organisms.

synthetic biology↗

bioRxiv2019

Batch cultivation of non-transgenic males suitable for SIT programs

The field performance of Sterile Insect Technique (SIT) is improved by sex-sorting and releasing only the sterile males. This can be accomplished by resource-intensive separation of males from females by morphology. Alternatively, sex-ratio biasing genetic constructs can be used to selectively kill one sex without the need for manual or automated sorting, but the resulting genetically engineered (GE) control agents would be subject to additional governmental regulation. Here we describe and demonstrate a method for the batch production of non-GE males that is applicable for sex-selective production of males suitable for genetic biocontrol programs. This method could be applied to generate the heterogametic sex (XY, or WZ) in any organism with chromosomal sex determination. We observed up to 100% sex-selection with batch cultures of more than 103 individuals. Using a stringent transgene detection assay, we demonstrate the potential of mass rearing of transgene free males.

synthetic biology↗

bioRxiv2018

Genetic incompatibility combined with female-lethality is effective and robust in simulations of Aedes aegypti population control

Recent reports of CRISPR/Cas9-based suppression gene drives in insects underscore the challenge of overcoming genetic resistance. Here we present results from agent-based simulation modeling of a novel Field-Amplified Male Sterility System (FAMSS) that outperforms suppression gene drives when challenged with genetic resistance. FAMSS combines a recently described synthetic genetic incompatibility approach with previously demonstrated female-lethality constructs. Our results suggest that FAMSS will be an effective strategy for temporally and spatially self-limited suppression of the disease vectoring mosquito, Aedes aegypti.

synthetic biology↗