Genetic Control Strategies Research Articles

Multi-omics analysis reveal the fall armyworm Spodoptera frugiperda tolerate high temperature by mediating chitin-related genes

Climate change facilitates the rapid invasion of agricultural pests, threatening global food security. The fall armyworm Spodoptera frugiperda is a highly polyphagous migratory pest tolerant to high temperatures, allowing its proliferation in harsh thermal environments. We aimed to demonstrate mechanisms of its high-temperature tolerance, particularly transcriptional and metabolic regulation, which are poorly understood. To achieve the aim, we examined the impact and mechanism of heat events on S. frugiperda by using multiple approaches: ecological measurements, transcriptomics, metabolomics, RNAi, and CRISPR/Cas9 technology. We observed that several physiological indices (larval survival rate, larval period, pupation rate, pupal weight, eclosion rate, and average fecundity) decreased as the temperature increased, with the 32 °C treatment displaying a significant difference from the control group at 26 °C. Significantly upregulated expression of genes encoding endochitinase and chitin deacetylase was observed in the chitin-binding, extracellular region, and carbohydrate metabolic process GO terms of hemolymph, fat body, and brain, exhibiting a tissue-specific pattern. Significantly enriched pathways (e.g., cutin, suberin, and wax biosynthesis; oxidative phosphorylation and cofactor biosynthesis; diverse amino acid biosynthesis and degradation; carbon metabolism; and energy metabolism), all of which are essential for S. frugiperda larvae to tolerate temperature, were found in metabolites that were expressed differently. Successful RNA interference targeting of the three chitin-related genes reduced gene expression levels and larval survival rate. Knockout of the endochitinase gene by using the CRISPR/Cas9 system significantly reduced the relative gene expression and increased sensitivity to high-temperature exposure. On the basis of our findings, theoretical foundations for understanding the high-temperature tolerance of S. frugiperda populations and latent genetic control strategies were established.

CRISPR-Cas9 and Cas12a target site richness reflects genomic diversity in natural populations of Anopheles gambiae and Aedes aegypti mosquitoes

Due to limitations in conventional disease vector control strategies including the rise of insecticide resistance in natural populations of mosquitoes, genetic control strategies using CRISPR gene drive systems have been under serious consideration. The identification of CRISPR target sites in mosquito populations is a key aspect for developing efficient genetic vector control strategies. While genome-wide Cas9 target sites have been explored in mosquitoes, a precise evaluation of target sites focused on coding sequence (CDS) is lacking. Additionally, target site polymorphisms have not been characterized for other nucleases such as Cas12a, which require a different DNA recognition site (PAM) and would expand the accessibility of mosquito genomes for genetic engineering. We undertook a comprehensive analysis of potential target sites for both Cas9 and Cas12a nucleases within the genomes of natural populations of Anopheles gambiae and Aedes aegypti from multiple continents. We demonstrate that using two nucleases increases the number of targets per gene. Also, we identified differences in nucleotide diversity between North American and African Aedes populations, impacting the abundance of good target sites with a minimal degree of polymorphisms that can affect the binding of gRNA. Lastly, we screened for gRNAs targeting sex-determination genes that could be widely applicable for developing field genetic control strategies. Overall, this work highlights the utility of employing both Cas9 and Cas12a nucleases and underscores the importance of designing universal genetic strategies adaptable to diverse mosquito populations.

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, which eliminates X-bearing sperm, and X-poisoning, which eliminates females 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 establish X-poisoning strains in the malaria vector Anopheles gambiae targeting three X-linked genes during spermatogenesis, resulting in male bias. We find that sex distortion is primarily driven by a loss of X-bearing sperm, with limited evidence for postzygotic lethality of female progeny. 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 explore 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.

Challenges of Robust RNAi-Mediated Gene Silencing in Aedes Mosquitoes.

In this study, we report the complexities and challenges associated with achieving robust RNA interference (RNAi)-mediated gene knockdown in the mosquitoes Aedes aegypti and Aedes albopictus, a pivotal approach for genetic analysis and vector control. Despite RNAi's potential for species-specific gene targeting, our independent efforts to establish oral delivery of RNAi for identifying genes critical for mosquito development and fitness encountered significant challenges, failing to reproduce previously reported potent RNAi effects. We independently evaluated a range of RNAi-inducing molecules (siRNAs, shRNAs, and dsRNAs) and administration methods (oral delivery, immersion, and microinjection) in three different laboratories. We also tested various mosquito strains and utilized microorganisms for RNA delivery. Our results reveal a pronounced inconsistency in RNAi efficacy, characterized by minimal effects on larval survival and gene expression levels in most instances despite strong published effects for the tested targets. One or multiple factors, including RNase activity in the gut, the cellular internalization and processing of RNA molecules, and the systemic dissemination of the RNAi signal, could be involved in this variability, all of which are barely understood in mosquitoes. The challenges identified in this study highlight the necessity for additional research into the underlying mechanisms of mosquito RNAi to develop more robust RNAi-based methodologies. Our findings emphasize the intricacies of RNAi application in mosquitoes, which present a substantial barrier to its utilization in genetic control strategies.

Integrated de novo transcriptome of Culex pipiens mosquito larvae as a resource for genetic control strategies

We present a de novo transcriptome of the mosquito vector Culex pipiens, assembled by sequences of susceptible and insecticide resistant larvae. The high quality of the assembly was confirmed by TransRate and BUSCO. A mapping percentage until 94.8% was obtained by aligning contigs to Nr, SwissProt, and TrEMBL, with 27,281 sequences that simultaneously mapped on the three databases. A total of 14,966 ORFs were also functionally annotated by using the eggNOG database. Among them, we identified ORF sequences of the main gene families involved in insecticide resistance. Therefore, this resource stands as a valuable reference for further studies of differential gene expression as well as to identify genes of interest for genetic-based control tools.

Development of a Genetic Algorithm-Based Control Strategy for Fuel Consumption Optimization in a Mild Hybrid Electrified Vehicle’s Electrified Propulsion System

Increasingly stringent pollutant emission regulations and a customer demand for a high-fuel economy drive the modern automotive industry to hurriedly solve the problem of decarbonization and powertrain efficiency, leading R&D towards alternative powertrain solutions and fuels. Electrification, today, plays the biggest role in the topic, with Mild Hybrid Electrified Vehicles (MHEVs) being the most cost-effective architectures, displaying dominance in smaller markets such as Brazil. One of the biggest challenges for HEVs’ development is the complexity of the hybrid control system, knowing when to actuate the electric machine, and the optimum power delivery, plus the gearshift schedule becomes a hard optimization problem that plays a key role in powertrain efficiency and cost savings for the customer. This paper proposes the implementation of a genetic algorithm (GA) as a machine learning-based control strategy to determine the torque split and the gear engaged for each driving condition of an MHEV operation, aiming to optimize fuel consumption. A quasi-static model of the vehicle was developed in Matlab/Simulink version 2022b, the virtual vehicle was then tested following the FTP75 and HWFET driving cycles. Simulation results indicate that the control decisions taken by the GA are qualitatively coherent for all operation conditions, and even quantitatively coherent in some cases, and that the software has the potential to be used as a control strategy outside the simulation environment, in future steps of development.

Innovations and Future Trends in Storage Pest Management

Current innovations and future trends in storage pest management, with a specific focus on the Indian context. The critical role of effective pest management in agriculture and food security is underscored, considering its impact on economic stability and public health. It begins by detailing the challenges faced in storage pest management, including the variety of pests like insects and rodents, and the damage they inflict on stored agricultural products. It critiques the limitations of traditional pest management methods, particularly chemical control, and highlights emerging issues such as climate change effects and pesticide resistance. The core examines the latest innovations in storage pest management. Advanced chemical approaches like novel pesticides and controlled release formulations are discussed, alongside the rise of nanotechnology applications in pest control, including nano-pesticides and smart delivery systems. The emergence of biological control innovations, particularly new biocontrol agents, and genetic control strategies like the sterile insect technique, marks a significant shift towards more sustainable pest management methods. Additionally, It explores developments in physical and mechanical control methods, emphasizing improved storage facilities and environmental control techniques. Integrated Pest Management (IPM) approaches are identified as a key future trend, promoting holistic and sustainable strategies, with case studies underscoring their success and practical application. It also addresses the vital role of policy and regulatory developments, considering the impact of global regulations and the importance of international cooperation and standards in shaping pest management practices in India. Ethical and environmental considerations form a crucial part of the discourse, focusing on the ecological impact of pest management and the balance between control measures and conservation efforts. It concludes with an examination of practical applications and case studies, providing insights into real-world challenges and the strategies employed to overcome them. Overall, It offers a thorough analysis of the state-of-the-art in storage pest management, highlighting the intersection of innovation, sustainability, and practicality. It aims to provide valuable guidance for researchers, policymakers, and practitioners in the field, contributing to the advancement of more effective, environmentally responsible, and sustainable pest management strategies in India.

RNAi-Mediated Silencing of Laccase 2 in Culex pipiens Pupae via Dehydration and Soaking Results in Multiple Defects in Cuticular Development.

Mosquitoes transmit a range of pathogens, causing devastating effects on human health. Population genetic control strategies have been developed and successfully used for several mosquito species. The most important step in identifying potential targets for mosquito control is the understanding of gene function. RNA interference (RNAi) is a powerful tool for gene silencing which has been widely used to study gene function in insects via knockdown of expression. The success of RNAi in insects depends on the efficient delivery of dsRNA into the cells, with microinjections being the most commonly used to study mosquito gene function. However, microinjections in the pupal stage lead to significant mortality in Aedes and Culex species, and few studies have performed microinjections in Culicinae pupae. Advanced techniques, such as CRISPR/Cas9 knockout, require establishing individual mosquito lines for each gene studied, and maintaining such lines may be limited by the insect-rearing capacity of a laboratory. Moreover, at times gene knockout during early development (embryo stage) has a deleterious effect on mosquito development, precluding the analysis of gene function in the pupal and adult stages and its potential for mosquito control. There is a need for a simple procedure that can be used for the fast and reliable examination of adult gene function via RNAi knockdown. Here, we focus on the aquatic stages of the mosquito life cycle and suggest a quick and easy assay for screening the functional role of genes in Culex pipiens mosquitoes without using microinjections. By dehydration of early stage pupae and subsequent rehydration in highly concentrated dsRNA, we achieved a moderate knockdown of laccase 2, a gene that turns on in the pupal stage and is responsible for melanization and sclerotization of the adult cuticle.

Embryonic microinjection of ribonucleoprotein complex (Cas9+sgRNA) of whitegene in melon fly, Zeugodacuscucurbitae (Coquillett) (Diptera: Tephritidae) produced whiteeye phenotype.

Melon fly, Zeugodacus cucurbitae (Coquillett) is a major pest of cucurbitaceous crops, and causes substantial yield losses and economic costs. CRISPR/Cas9 is a rapid and effective site-specific genome editing tool for the generation of genetic changes that are stable and heritable. The CRISPR/Cas9 tool uses synthetically designed single guide RNA (sgRNA) that is complementary to the target gene and guides the Cas9 enzyme to perform nuclease activity by making double-strand breaks in the target DNA sequences. This tool can be effectively exploited to improve traits critical for the management of insect pests by targeting specific genes encoding these traits without the need of extensive genetic information. The white gene is an important gene responsible for the transport of body pigment precursor molecules. In this study, we produced effective mutagenesis of the white gene of Z. cucurbitae using the CRISPR/Cas9 tool with double sgRNAto target multiple sites of white to increase the efficiency in the generation of frame-shift mutations resulting in the white eye phenotype in adults. This was achieved through embryonic microinjection of the ribonucleoprotein (RNP) complex in the pre-blastoderm embryo stage 1 h after embryo laying. Our success with the production of a white eye mutant fly by CRISPR/Cas9 mutagenesis is important for the research on gene function and protein-level modifications in melon fly and forms the basis for the development of new genetic control strategies such as precision guided sterile insect technique (pgSIT) for this pest of economic significance.

Optimizing CRE and PhiC31 mediated recombination in Aedes aegypti.

Introduction: 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. Methods: Here we present two improved methods for genetic manipulation in this species. Use of transgenic lines which express Cre recombinase and a plasmid-based method for expressing PhiC31 when injected into early embryos. Results: 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. Discussion: Here we have expanded the toolbox for synthetic biology in Ae. aegypti. These methods can be easily transferred into other mosquito and even insect species by identifying appropriate promoter sequences. This advances the ability to manipulate these insects for fundamental studies, and for more applied approaches for pest control.

Measuring the Impact of Genetic Heterogeneity and Chromosomal Inversions on the Efficacy of CRISPR-Cas9 Gene Drives in Different Strains of Anopheles gambiae.

The human malaria vector Anopheles gambiae is becoming increasingly resistant to insecticides, spurring the development of genetic control strategies. CRISPR-Cas9 gene drives can modify a population by creating double-stranded breaks at highly specific targets, triggering copying of the gene drive into the cut site ("homing"), ensuring its inheritance. The DNA repair mechanism responsible requires homology between the donor and recipient chromosomes, presenting challenges for the invasion of laboratory-developed gene drives into wild populations of target species An. gambiae species complex, which show high levels of genome variation. Two gene drives (vas2-5958 and zpg-7280) were introduced into three An. gambiae strains collected across Africa with 5.3-6.6% variation around the target sites, and the effect of this variation on homing was measured. Gene drive homing across different karyotypes of the 2La chromosomal inversion was also assessed. No decrease in gene drive homing was seen despite target site heterology, demonstrating the applicability of gene drives to wild populations.

Single-cell profiling of Anopheles gambiae spermatogenesis defines the onset of meiotic silencing and premeiotic overexpression of the X chromosome

Understanding development and genetic regulation in the Anopheles gambiae germline is essential to engineer effective genetic control strategies targeting this malaria mosquito vector. These include targeting the germline to induce sterility or using regulatory sequences to drive transgene expression for applications such as gene drive. However, only very few germline-specific regulatory elements have been characterised with the majority showing leaky expression. This has been shown to considerably reduce the efficiency of current genetic control strategies, which rely on regulatory elements with more tightly restricted spatial and/or temporal expression. Meiotic silencing of the sex chromosomes limits the flexibility of transgene expression to develop effective sex-linked genetic control strategies. Here, we build on our previous study, dissecting gametogenesis into four distinct cell populations, using single-cell RNA sequencing to define eight distinct cell clusters and associated germline cell–types using available marker genes. We reveal overexpression of X-linked genes in a distinct cluster of pre-meiotic cells and document the onset of meiotic silencing of the X chromosome in a subcluster of cells in the latter stages of spermatogenesis. This study provides a comprehensive dataset, characterising the expression of distinct cell types through spermatogenesis and widening the toolkit for genetic control of malaria mosquitoes.

Viral Protein 1 (VP1) Sequence-Based Genetic Diversity of SAT 2 FMDV Circulating in Ethiopia from 1990 to 2015.

Pathogen molecular epidemiology determines the origin of specific outbreaks locality of foot-and-mouth disease virus serotype South African Territories-2 sequence-based analysis of highly variable Viral Protein 1 (VP1), which helps to identify the evolution of this virus through time and space. The objective of this study was to compare the differences between SAT-2 VP1 sequences of FMDV circulated in Ethiopia from 1990 to 2015 at the genetic level. The nucleotide and amino acid sequences were analyzed using Basic Local Alignment Search Tools (BLAST), Multiple sequence alignment and sequence editing and Phylogenetic tree reconstruction. The nucleotide and amino acid sequences alignment, distance matrix, and phylogenetic tree constructions were done using the MEGA 6.0 software package. In this analysis, we found 76% nucleotide identities and amino acid similarities among the sequences. The overall group mean distance at nucleotide level was 19% with a mean intra-population diversity of 2%. The lowest sequence variation was observed among sequences obtained from the years 2007/09/10, 2014/15, and 1990/91 which was less than 5% among them. This analysis revealed that in the last 25 years, four different topotypes of the FMDV SAT-2 were circulating in Ethiopia. The Arg-Gly-Asp (RGD) amino acid (AA) motif at AA position 144-146 within the G-H loop of the VP1 protein of FMDV is conserved, but up- and downstream hyper-variable AA sequences are identified. In this study, it was observed that four topotypes (IV, XIV, XIII, and VII) were circulating in Ethiopia for 25 years. Further, compared with sequences from neighboring countries (Sudan, Kenya) confirmed the presence of these topotypes. Pertinent to this genetic diversity control strategies in Ethiopia should be based on having regular antigenic and genetic vaccine matching tests with the circulating strain within a defined period, space, transboundary nature of the disease and applying biosecurity measures.

Genome Assembly and Population Sequencing Reveal Three Populations and Signatures of Insecticide Resistance of Tuta absoluta in Latin America.

Tuta absoluta is one of the largest threats to tomato agriculture worldwide. Native to South America, it has rapidly spread throughout Europe, Africa, and Asia over the past two decades. To understand how T. absoluta has been so successful and to improve containment strategies, high-quality genomic resources and an understanding of population history are critical. Here, we describe a highly contiguous annotated genome assembly, as well as a genome-wide population analysis of samples collected across Latin America. The new genome assembly has an L50 of 17 with only 132 contigs. Based on hundreds of thousands of single nucleotide polymorphisms, we detect three major population clusters in Latin America with some evidence of admixture along the Andes Mountain range. Based on coalescent simulations, we find these clusters diverged from each other tens of thousands of generations ago prior to domestication of tomatoes. We further identify several genomic loci with patterns consistent with positive selection and that are related to insecticide resistance, immunity, and metabolism. This data will further future research toward genetic control strategies and inform future containment policies.

CRISPR-based genetic control strategies for insect pests

Genetic control strategies such as the sterile insect technique have successfully fought insect pests worldwide. The CRISPR (clustered regularly interspaced short palindromic repeats) technology, together with high-quality genomic resources obtained in more and more species, greatly facilitates the development of novel genetic control insect strains that can be used in area-wide and species-specific pest control programs. Here, we review the research progress towards state-of-art CRISPR-based genetic control strategies, including gene drive, sex ratio distortion, CRISPR-engineered genetic sexing strains, and precision-guided sterile insect technique. These strategies' working mechanisms, potential resistance development mechanisms, and regulations are illustrated and discussed. In addition, recent developments such as stacked and conditional systems are introduced. We envision that the advances in genetic technology will continue to be one of the driving forces for developing the next generation of pest control strategies.

Effects of antibiotics on the in vitro expression of tetracycline-off constructs and the performance of Drosophila suzukii female-killing strains.

Genetic control strategies such as the Release of Insects Carrying a Dominant Lethal (RIDL) gene and Transgenic Embryonic Sexing System (TESS) have been demonstrated in the laboratory and/or deployed in the field. These strategies are based on tetracycline-off (Tet-off) systems which are regulated by antibiotics such as Tet and doxycycline (Dox). Here, we generated several Tet-off constructs carrying a reporter gene cassette mediated by a 2A peptide. Different concentrations (0.1, 10, 100, 500, and 1,000μg/mL) and types (Tet or Dox) of antibiotics were used to evaluate their effects on the expression of the Tet-off constructs in the Drosophila S2 cells. One or both of the two concentrations, 100 and 250μg/mL, of Tet or Dox were used to check the influence on the performances of a Drosophila suzukii wild-type strain and female-killing (FK) strains employing TESS. Specifically, the Tet-off construct for these FK strains contains a Drosophila suzukii nullo promoter to regulate the tetracycline transactivator gene and a sex-specifically spliced pro-apoptotic gene hid Ala4 to eliminate females. The results suggested that the in vitro expression of the Tet-off constructs was controlled by antibiotics in a dose-dependent manner. ELISA experiments were carried out identifying Tet at 34.8ng/g in adult females that fed on food supplemented with Tet at 100μg/mL. However, such method did not detect Tet in the eggs produced by antibiotic-treated flies. Additionally, feeding Tet to the parents showed negative impact on the fly development but not the survival in the next generation. Importantly, we demonstrated that under certain antibiotic treatments females could survive in the FK strains with different transgene activities. For the strain V229_M4f1 which showed moderate transgene activity, feeding Dox to fathers or mothers suppressed the female lethality in the next generation and feeding Tet or Dox to mothers generated long-lived female survivors. For the strain V229_M8f2 which showed weak transgene activity, feeding Tet to mothers delayed the female lethality for one generation. Therefore, for genetic control strategies employing the Tet-off system, the parental and transgenerational effects of antibiotics on the engineered lethality and insect fitness must be carefully evaluated for a safe and efficient control program.

Evaluating the Mating Competency of Genetically Modified Male Mosquitoes in Laboratory Conditions.

Efforts to eradicate mosquito-borne diseases have increased the demand for genetic control strategies, many of which involve the release of genetically modified (GM) mosquito males into natural populations. The first hurdle for GM males is to compete with their wild-type counterparts for access to females. Here, we introduce an eye color-based mating assay, in which both Lvp wild-type and kynurenine 3-monooxygenase (kmo)-null males compete for access to kmo-null females, and therefore the eye color phenotype (black or white) of the progeny is dependent on the parental mating pair. A series of tests addressed that male mating competitiveness between the two strains can significantly be influenced by adult density, light intensity, and mating duration. Interestingly, the mating competitiveness of males was not correlated with body size, which was negatively influenced by a high larval density. Lastly, this eye color-associated assay was applied to characterize GM mosquitoes in their mating competitiveness, establishing this method as a fast and precise way of benchmarking this fitness parameter for laboratory-raised males.

Research on the optimization strategy of lithium battery electrode correction system based on algorithm fusion

In the control system of a lithium battery rolling mill, the correction system was crucial. This was because the correction system had a significant impact on the performance of the lithium battery rolling mill, including high precision and efficient rolling quality. However, the non-linearity of the correction system and the uncertainty of the correction system made it a challenging problem to achieve a high precision correction control. The contribution and innovation of this paper was a genetic fuzzy PID control strategy based on Kalman filter, which was proposed and applied to the control of lithium battery rolling mill correction technology. In order to achieve intelligent control of a high-precision electrode rolling mill correction system, an algorithm fusion control scheme was proposed. Firstly, a novel and detailed correction system model was presented. Next, the initial PID parameters of the correction were optimized by means of a genetic algorithm so that the PID parameters could be adapted to the correction control process and then optimized again by adding an extended Kalman filter. Finally, the lithium battery rolling mill correction control system was validated, tested and commissioned in the field. The results showed that the designed algorithm could meet the working requirements of the lithium battery rolling mill and that it improved the accuracy of the correction system. In the actual lithium battery rolling mill production process, the algorithm was compared with a conventional PID. Compared with the common single algorithm, the fusion algorithm proposed in this paper was a complete set of high precision correction control system algorithm to solve the high precision problem faced by the correction system in the actual lithium battery rolling mill correction system.

CRISPR-Mediated Cassette Exchange (CriMCE): A Method to Introduce and Isolate Precise Marker-Less Edits.

The introduction of small unmarked edits to the genome of insects is essential to study the molecular underpinnings of important biological traits, such as resistance to insecticides and genetic control strategies. Advances in CRISPR genome engineering have made this possible, but prohibitively laborious for most laboratories due to low rates of editing and the lack of a selectable marker. To facilitate the generation and isolation of precise marker-less edits we have developed a two-step method based on CRISPR-mediated cassette exchange (CriMCE) of a marked placeholder for a variant of interest. This strategy can be used to introduce a wider range of potential edits compared with previous approaches while consolidating the workflow. We present proof-of-principle that CriMCE is a powerful tool by engineering three single nucleotide polymorphism variants into the genome of Anopheles gambiae, with 5-41 × higher rates of editing than homology-directed repair or prime editing.

Competitive Sperm-Marked Beetles for Monitoring Approaches in Genetic Biocontrol and Studies in Reproductive Biology.

Sperm marking provides a key tool for reproductive biology studies, but it also represents a valuable monitoring tool for genetic pest control strategies such as the sterile insect technique. Sperm-marked lines can be generated by introducing transgenes that mediate the expression of fluorescent proteins during spermatogenesis. The homozygous lines established by transgenesis approaches are going through a genetic bottleneck that can lead to reduced fitness. Transgenic SIT approaches have mostly focused on Dipteran and Lepidopteran pests so far. With this study, we provide sperm-marked lines for the Coleopteran pest model organism, the red flour beetle Tribolium castaneum, based on the β2-tubulin promoter/enhancer driving red (DsRed) or green (EGFP) fluorescence. The obtained lines are reasonably competitive and were thus used for our studies on reproductive biology, confirming the phenomenon of ‘last-male sperm precedence’ and that the spermathecae are deployed for long-term sperm storage, enabling the use of sperm from first mating events even after secondary mating events for a long period of time. The homozygosity and competitiveness of the lines will enable future studies to analyze the controlled process of sperm movement into the long-term storage organ as part of a post-mating cryptic female choice mechanism of this extremely promiscuous species.