CYP4CS5-mediated thiamethoxam and clothianidin resistance is accompanied by fitness cost in the whitefly Bemisia tabaci.

BACKGROUND: Understanding the trade-offs between insecticide resistance and the associated fitness is of particular importance to sustainable pest control. One of the most devastating pest worldwide, the whitefly Bemisia tabaci, has developed resistance to various insecticides, especially the neonicotinoid group. Although neonicotinoid resistance often is conferred by P450s-mediated metabolic resistance, the relationship between such resistance and the associated fitness phenotype remains largely elusive. By gene cloning, quantitative reverse transcription (qRT)-PCR, RNA interference (RNAi), transgenic Drosophila melanogaster, metabolism capacity in vitro and 'two sex-age stage' life table study, this study aims to explore the molecular role of a P450 gene CYP4CS5 in neonicotinoid resistance and to investigate whether such resistance mechanism carries fitness costs in the whitefly. RESULTS: Our bioassay tests showed that a total of 13 field-collected populations of B. tabaci MED biotype displayed low-to-moderate resistance to thiamethoxam and clothianidin. Compared to the laboratory susceptible strain, we then found that an important P450 CYP4CS5 was remarkably upregulated in the field resistant populations. Such overexpression of CYP4CS5 had a good match with the resistance level among the whitefly samples. Further exposure to the two neonicotinoids resulted in an increase in CYP4CS5 expression. These results implicate that overexpression of CYP4CS5 is closely correlated with thiamethoxam and clothianidin resistance. RNAi knockdown of CYP4CS5 increased mortality of the resistant and susceptible populations after treatment with thiamethoxam and clothianidin in bioassay, but obtained an opposite result when using a transgenic line of D. melanogaster expressing CYP4CS5. Metabolic assays in vitro revealed that CYP4CS5 exhibited certain capacity of metabolizing thiamethoxam and clothianidin. These in vivo and in vitro assays indicate an essential role of CYP4CS5 in conferring thiamethoxam and clothianidin resistance in whitefly. Additionally, our life-table analysis demonstrate that the field resistant whitefly exhibited a prolonged development time, shortened longevity and reduced fecundity compared to the susceptible, suggesting an existing fitness cost as a result of the resistance. CONCLUSION: Collectively, in addition to the important role of CYP4CS5 in conferring thiamethoxam and clothianidin resistance, this resistance mechanism is associated with fitness costs in the whitefly. These findings not only contribute to the development of neonicotinoids resistance management strategies, but also provide a new target for sustainable whitefly control. © 2023 Society of Chemical Industry.

Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes.

Transgenic mosquitoes often display fitness costs compared to their wild-type counterparts. In this regard, fitness cost studies involve collecting life parameter data from genetically modified mosquitoes and comparing them to mosquitoes lacking transgenes from the same genetic background. This manuscript illustrates how to measure common life history traits in the mosquito Aedes aegypti, including fecundity, wing size and shape, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These parameters were chosen because they reflect reproductive success, are simple to measure, and are commonly reported in the literature. The representative results quantify fitness costs associated with either a gene knock-out or a single insertion of a gene drive element. Standardizing how life parameter data are collected is important because such data may be used to compare the health of transgenic mosquitoes generated across studies or to model the transgene fixation rate in a simulated wild-type mosquito population. Although this protocol is specific for transgenic Aedes aegypti, the protocol may also be used for other mosquito species or other experimental treatment conditions, with the caveat that certain biological contexts may require special adaptations.

Large-scale and cost-effective production of recombinant human serum albumin (rHSA) in transgenic Bombyx mori cocoons.

HSA is considered a versatile natural cargo carrier with multiple bio-functions and applications. However, insufficient supply of HSA has limited widespread use. Although various recombinant expression systems had been applied to produce the rHSA to overcome the limited resource, cost-effective and large scale production of rHSA remains a challenge. Herein, we provide a strategy for the large-scale and cost-effective production of rHSA in cocoons of transgenic silkworms, achieving a final 13.54 ± 1.34 g/kg of rHSA yield in cocoons. rHSA was efficiently synthesized and stable over the long-term in the cocoons at room temperature. Artificial control of silk crystal structure during silk spinning significantly facilitated rHSA extraction and purification, with 99.69 ± 0.33 % purity and a productivity of 8.06 ± 0.17 g rHSA from 1 kg cocoons. The rHSA had the same secondary structure to natural HSA, along with effective drug binding capacity, biocompatibility, and bio-safe. The rHSA was successfully evaluated as a potential substitute in serum-free cell culture. These findings suggest the silkworm bioreactor is promising for large-scale and cost-effective production of high quality rHSA to meet the increased worldwide demand.

Safety assessment of subchronic feeding of insect-resistant and herbicide-resistant transgenic soybeans to juvenile channel catfish (Ictalurus punctatus).

Transgenic soybean is one of the most planted crops for human food and animal feed. The channel catfish (Ictalurus punctatus) is an important aquatic organism cultured worldwide. In this study, the effect of six different soybean diets containing: two transgenic soybeans expressing different types of cp4-epsps, Vip3Aa and pat genes (DBN9004 and DBN8002), their non-transgenic parent JACK, and three conventional soybean varieties (Dongsheng3, Dongsheng7, and Dongsheng9) was investigated in juvenile channel catfish for eight weeks, and a safety assessment was performed. During the experiment, no difference in survival rate was observed in six groups. The hepatosomatic index (HSI) and condition factor (CF) showed no significant difference. Moreover, comparable feed conversion (FC), feeding rate (FR), and feed conversion ratio (FCR) were found between transgenic soybean and JACK groups. Assessment of growth performance showed that the weight gain rate (WGR) and specific growth rate (SGR) of channel catfish were consistent. In addition, there were no changes in enzyme activity indexes (lactate dehydrogenase (LDH), total antioxidant capacity (T-AOC), aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) in channel catfish among treatments. The research provided an experimental basis for the aquaculture feed industry to employ transgenic soybean DBN9004 and DBN8002 for commercial purposes.

Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk.

Heart disease is the leading cause of morbidity and mortality worldwide. Due to their low cost, ease of handling, and abundance of transgenic strains, rodents have become essential models for cardiovascular research. However, spontaneous lethal cardiac arrhythmias that often cause mortality in heart disease patients are rare in rodent models of heart disease. This is primarily due to the species differences in cardiac electrical properties between human and rodents and poses a challenge to the study of cardiac arrhythmias using rodents. This protocol describes an approach to enable efficient transgene expression in mouse and rat ventricular myocardium using echocardiography-guided intramuscular injections of recombinant virus (adenovirus and adeno-associated virus). This work also outlines a method to enable reliable assessment of cardiac susceptibility to arrhythmias using isolated, Langendorff-perfused mouse and rat hearts with both adrenergic and programmed electrical stimulations. These techniques are critical for studying heart rhythm disorders associated with adverse cardiac remodeling after injuries, such as myocardial infarction.

Inducible and tissue-specific cell labeling in Cre-ERT2 transgenic Xenopus lines.

Investigating cell lineage requires genetic tools that label cells in a temporal and tissue-specific manner. The bacteriophage-derived Cre-ERT2 /loxP system has been developed as a genetic tool for lineage tracing in many organisms. We recently reported a stable transgenic Xenopus line with a Cre-ERT2 /loxP system driven by the mouse Prrx1 (mPrrx1) enhancer to trace limb fibroblasts during the regeneration process (Prrx1:CreER line). Here we describe the detailed technological development and characterization of such line. Transgenic lines carrying a CAG promoter-driven Cre-ERT2 /loxP system showed conditional labeling of muscle, epidermal, and interstitial cells in both the tadpole tail and the froglet leg upon 4-hydroxytamoxifen (4OHT) treatment. We further improved the labeling efficiency in the Prrx1:CreER lines from 12.0% to 32.9% using the optimized 4OHT treatment regime. Careful histological examination showed that Prrx1:CreER lines also sparsely labeled cells in the brain, spinal cord, head dermis, and fibroblasts in the tail. This work provides the first demonstration of conditional, tissue-specific cell labeling with the Cre-ERT2 /loxP system in stable transgenic Xenopus lines.

[The in vivo imaging of zebrafish for chemical vascular toxicity assessment].

Objective: To evaluate the vascular toxicity of chemicals by a real-time observation approach using the transgenic zebrafish. Methods: The spatiotemporal vascular alterations of transgenic zebrafish after chemical exposure were assessed by laser confocal microscopy and high-content screening analysis, respectively. Results: The method using Laser Confocal Microscopy (LCM) is easier to operate and yields high-resolution images, while it is lower throughput and inefficient. In contrast, high-content analysis (HCA) analysis obtains high-quality data of vascular toxicity manifesting whole blood vasculature, whereas it requires delicate operation procedures and advanced experimental conditions. Conclusion: Two kinds of zebrafish imaging methods each have advantages and disadvantages. LCM is suitable for the evaluation of a small number of chemicals. HCA, a cutting-edge technology, has great potential for chemical safety assessment allowing high throughput vascular toxicity tests of a good number of chemicals at a time.

Quantitative spatial and temporal assessment of regulatory element activity in zebrafish.

Mutations or genetic variation in noncoding regions of the genome harbouring cis-regulatory elements (CREs), or enhancers, have been widely implicated in human disease and disease risk. However, our ability to assay the impact of these DNA sequence changes on enhancer activity is currently very limited because of the need to assay these elements in an appropriate biological context. Here, we describe a method for simultaneous quantitative assessment of the spatial and temporal activity of wild-type and disease-associated mutant human CRE alleles using live imaging in zebrafish embryonic development. We generated transgenic lines harbouring a dual-CRE dual-reporter cassette in a pre-defined neutral docking site in the zebrafish genome. The activity of each CRE allele is reported via expression of a specific fluorescent reporter, allowing simultaneous visualisation of where and when in development the wild-type allele is active and how this activity is altered by mutation.

Assessment and Validation of Globodera pallida as a Novel In Vivo Model for Studying Alzheimer's Disease.

BACKGROUND: Whole transgenic or non-transgenic organism model systems allow the screening of pharmacological compounds for protective actions in Alzheimer's disease (AD). AIM: In this study, a plant parasitic nematode, Globodera pallida, which assimilates intact peptides from the external environment, was investigated as a new potential non-transgenic model system of AD. Methods: Fresh second-stage juveniles of G. pallida were used to measure their chemosensory, perform immunocytochemistry on their neurological structures, evaluate their survival rate, measure reactive oxygen species, and determine total oxidized glutathione to reduced glutathione ratio (GSSG/GSH) levels, before and after treatment with 100 µM of various amyloid beta (Aß) peptides (1-40, 1-42, 17-42, 17-40, 1-28, or 1-16). Wild-type N2 C. elegans (strain N2) was cultured on Nematode Growth Medium and directly used, as control, for chemosensory assays. RESULTS: We demonstrated that: (i) G. pallida (unlike Caenorhabditis elegans) assimilates amyloid-ß (Aß) peptides which co-localise with its neurological structures; (ii) pre-treatment with various Aß isoforms (1-40, 1-42, 17-42, 17-40, 1-28, or 1-16) impairs G. pallida's chemotaxis to differing extents; (iii) Aß peptides reduced survival, increased the production of ROS, and increased GSSG/GSH levels in this model; (iv) this unique model can distinguish differences between different treatment concentrations, durations, and modalities, displaying good sensitivity; (v) clinically approved neuroprotective agents were effective in protecting G. pallida from Aß (1-42) exposure. Taken together, the data indicate that G. pallida is an interesting in vivo model with strong potential for discovery of novel bioactive compounds with anti-AD activity.

A deterministic genotyping workflow reduces waste of transgenic individuals by two-thirds.

We present a deterministic workflow for genotyping single and double transgenic individuals directly upon nascence that prevents overproduction and reduces wasted animals by two-thirds. In our vector concepts, transgenes are accompanied by two of four clearly distinguishable transformation markers that are embedded in interweaved, but incompatible Lox site pairs. Following Cre-mediated recombination, the genotypes of single and double transgenic individuals were successfully identified by specific marker combinations in 461 scorings.

Assessment of heterogeneity in collective endothelial cell behavior with multicolor clonal cell tracking to predict arteriovenous remodeling.

Arteries and veins form in a stepwise process that combines vasculogenesis and sprouting angiogenesis. Despite extensive data on the mechanisms governing blood vessel assembly at the single-cell level, little is known about how collective cell migration contributes to the organization of the balanced distribution between arteries and veins. Here, we use an endothelial-specific zebrafish reporter, arteriobow, to label small cohorts of arterial cells and trace their progeny from early vasculogenesis throughout arteriovenous remodeling. We reveal that the genesis of arteries and veins relies on the coordination of 10 types of collective cell dynamics. Within these behavioral categories, we identify a heterogeneity of collective cell motion specific to either arterial or venous remodeling. Using pharmacological blockade, we further show that cell-intrinsic Notch signaling and cell-extrinsic blood flow act as regulators in maintaining the heterogeneity of collective endothelial cell behavior, which, in turn, instructs the future territory of arteriovenous remodeling.

In vitro assessment of antitumor immune responses using tumor antigen proteins produced by transgenic silkworms.

The evaluation of antitumor immune responses is essential for immune monitoring to predict clinical outcomes as well as treatment efficacies in cancer patients. In this study, we produced two tumor antigen (TA) proteins, melanoma antigen family A4 and wild type p53, using TG silkworm systems and evaluated anti-TA-specific immune responses by enzyme-linked immunosorbent spot assays in patients with head and neck cancer. Eleven (61.1%) of 18 patients showed significant IFN-γ production in response to at least one TA; however, the presence of TA-specific immune responses did not significantly contribute to better prognosis (overall survival, p = 0.1768; progression-free survival, p = 0.4507). Further studies will need to be performed on a larger scale to better assess the clinical significance of these systems. The production of multiple TA proteins may provide new avenues for the development of immunotherapeutic strategies to stimulate a potent and specific immune response against tumor cells as well as precise assessment of antitumor immune responses in cancer patients.

Assessment of Developmental and Reproductive Fitness of Dengue-Resistant Transgenic Aedes aegypti and Improvement of Fitness Using Antibiotics.

BACKGROUND: Genetic modification offers opportunities to introduce artificially created molecular defence mechanisms to vector mosquitoes to counter diseases causing pathogens such as the dengue virus, malaria parasite, and Zika virus. RNA interference is such a molecular defence mechanism that could be used for this purpose to block the transmission of pathogens among human and animal populations. In our previous study, we engineered a dengue-resistant transgenic Ae. aegypti using RNAi to turn off the expression of dengue virus serotype genomes to reduce virus transmission, requiring assessment of the fitness of this mosquito with respect to its wild counterpart in the laboratory and semifield conditions. METHOD: Developmental and reproductive fitness parameters of TM and WM have assessed under the Arthropod Containment Level 2 conditions, and the antibiotic treatment assays were conducted using co-trimoxazole, amoxicillin, and doxycycline to assess the developmental and reproductive fitness parameters. RESULTS: A significant reduction of developmental and reproductive fitness parameters was observed in transgenic mosquito compared to wild mosquitoes. However, it was seen in laboratory-scale studies that the fitness of this mosquito has improved significantly in the presence of antibiotics such as co-trimoxazole, amoxicillin, and doxycycline in their feed. CONCLUSION: Our data indicate that the transgenic mosquito produced had a reduction of the fitness parameters and it may lead to a subsequent reduction of transgenic vector density over the generations in field applications. However, antibiotics of co-trimoxazole, amoxicillin, and doxycycline have shown the improvement of fitness parameters indicating the usefulness in field release of transgenic mosquitoes.

Platforms for Personalized Polytherapeutics Discovery in COVID-19.

The COVID-19 pandemic entered its third and most intense to date wave of infections in November 2020. This perspective article describes how combination therapies (polytherapeutics) are a needed focus for helping battle the severity of complications from SARS-CoV-2 infection. It outlines the types of systems that are needed for fast and efficient combinatorial assessment of therapeutic candidates. Proposed are micro-physiological systems using human iPSC as a format for tissue-specific modeling of infection, the use of gene-humanized zebrafish and C. elegans for combinatorial drug screens due to the animals being addressable in liquid multi-well formats, and the use of engineered pseudo-typing systems to safely model infection in the transgenic animals and engineered tissue systems.

Transgenic cotton and sterile insect releases synergize eradication of pink bollworm a century after it invaded the United States.

Invasive organisms pose a global threat and are exceptionally difficult to eradicate after they become abundant in their new habitats. We report a successful multitactic strategy for combating the pink bollworm (Pectinophora gossypiella), one of the world's most invasive pests. A coordinated program in the southwestern United States and northern Mexico included releases of billions of sterile pink bollworm moths from airplanes and planting of cotton engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). An analysis of computer simulations and 21 y of field data from Arizona demonstrate that the transgenic Bt cotton and sterile insect releases interacted synergistically to reduce the pest's population size. In Arizona, the program started in 2006 and decreased the pest's estimated statewide population size from over 2 billion in 2005 to zero in 2013. Complementary regional efforts eradicated this pest throughout the cotton-growing areas of the continental United States and northern Mexico a century after it had invaded both countries. The removal of this pest saved farmers in the United States $192 million from 2014 to 2019. It also eliminated the environmental and safety hazards associated with insecticide sprays that had previously targeted the pink bollworm and facilitated an 82% reduction in insecticides used against all cotton pests in Arizona. The economic and social benefits achieved demonstrate the advantages of using agricultural biotechnology in concert with classical pest control tactics.

Genetic Engineering of Livestock: The Opportunity Cost of Regulatory Delay.

Genetically engineered (GE) livestock were first reported in 1985, and yet only a single GE food animal, the fast-growing AquAdvantage salmon, has been commercialized. There are myriad interconnected reasons for the slow progress in this once-promising field, including technical issues, the structure of livestock industries, lack of public research funding and investment, regulatory obstacles, and concern about public opinion. This review focuses on GE livestock that have been produced and documents the difficulties that researchers and developers have encountered en route. Additionally, the costs associated with delayed commercialization of GE livestock were modeled using three case studies: GE mastitis-resistant dairy cattle, genome-edited porcine reproductive and respiratory syndrome virus-resistant pigs, and the AquAdvantage salmon. Delays of 5 or 10 years in the commercialization of GE livestock beyond the normative 10-year GE product evaluation period were associated with billions of dollars in opportunity costs and reduced global food security.

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans.

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and degradation instigates a multitude of age-related diseases. Decline of proteostasis quality control mechanisms is a hallmark of ageing. Biochemical methods to detect de novo protein synthesis are still limited, have several disadvantages and cannot be performed in live cells or animals. Caenorhabditis elegans, being transparent and easily genetically modified, is an excellent model to monitor protein synthesis rates by using imaging techniques. Here, we introduce and describe a method to measure de novo protein synthesis in vivo utilizing fluorescence recovery after photobleaching (FRAP). Transgenic animals expressing fluorescent proteins in specific cells or tissues are irradiated by a powerful light source resulting in fluorescence photobleaching. In turn, assessment of fluorescence recovery signifies new protein synthesis in cells and/or tissues of interest. Hence, the combination of transgenic nematodes, genetic and/or pharmacological interventions together with live imaging of protein synthesis rates can shed light on mechanisms mediating age-dependent proteostasis collapse.

DNA methylation study of Huntington's disease and motor progression in patients and in animal models.

Although Huntington's disease (HD) is a well studied Mendelian genetic disorder, less is known about its associated epigenetic changes. Here, we characterize DNA methylation levels in six different tissues from 3 species: a mouse huntingtin (Htt) gene knock-in model, a transgenic HTT sheep model, and humans. Our epigenome-wide association study (EWAS) of human blood reveals that HD mutation status is significantly (p < 10-7) associated with 33 CpG sites, including the HTT gene (p = 6.5 × 10-26). These Htt/HTT associations were replicated in the Q175 Htt knock-in mouse model (p = 6.0 × 10-8) and in the transgenic sheep model (p = 2.4 × 10-88). We define a measure of HD motor score progression among manifest HD cases based on multiple clinical assessments. EWAS of motor progression in manifest HD cases exhibits significant (p < 10-7) associations with methylation levels at three loci: near PEX14 (p = 9.3 × 10-9), GRIK4 (p = 3.0 × 10-8), and COX4I2 (p = 6.5 × 10-8). We conclude that HD is accompanied by profound changes of DNA methylation levels in three mammalian species.

Diet type influences the gut microbiome and nutrient assimilation of Genetically Improved Farmed Tilapia (Oreochromis niloticus).

Nile tilapia, Oreochromis niloticus is the third most commonly farmed finfish species in the world, accounting for nearly 5% of global aquaculture production. In the past few decades much of the success of this species has been attributed to the development and distribution of Genetically Improved Farmed Tilapia (GIFT). Despite the increasing availability of GIFT, the productivity of small-scale farming remains highly variable, particularly in developing nations. Commercial fish-feed pellets can increase fish farm productivity; however, many small-scale farmers rely on other means of feeding fish due to the high cost and limited availability of commercial fish feed pellets. Therefore, understanding how locally-sourced feeds affect the production of GIFT is an important step towards improving feeding practices, particularly for farmers with low financial capital. This study used stable isotope analysis (SIA) and 16S rRNA gene sequencing to compare the effects of a locally-sourced vegetable-based diet and commercial pellet-based diets on the relative condition, nutrient assimilation patterns and gastrointestinal microbiota of GIFT. GIFT fed a locally-sourced diet were smaller, and in a significantly poorer condition than those fed with commercial fish feeds. SIA showed no differences in dietary carbon between the two diets; however, δ13C, poor fish condition and the abundance of specific bacterial taxa (of such as Fusobacteria) were correlated. SIA revealed that GIFT fed locally-sourced diets that predominantly consisted of vegetables were significantly enriched in δ15N despite a perceived lack of dietary protein. This enrichment suggests that GIFT fed a locally-sourced diet may be supplementing their diet via cannibalism, a behaviour representative of poor farming practice. Overall this study highlights the need to increase the availability of suitable GIFT feeds in developing nations. The development a low-cost feed alternative could improve the success of small-scale GIFT farmers in PNG, increasing both food and income security within the region.

Evaluation of the CRISPR/Cas9 Genetic Constructs in Efficient Disruption of Porcine Genes for Xenotransplantation Purposes Along with an Assessment of the Off-Target Mutation Formation.

The increasing life expectancy of humans has led to an increase in the number of patients with chronic diseases and organ failure. However, the imbalance between the supply and the demand for human organs is a serious problem in modern transplantology. One of many solutions to overcome this problem is the use of xenotransplantation. The domestic pig (Sus scrofa domestica) is currently considered as the most suitable for human organ procurement. However, there are discrepancies between pigs and humans that lead to the creation of immunological barriers preventing the direct xenograft. The introduction of appropriate modifications to the pig genome to prevent xenograft rejection is crucial in xenotransplantation studies. In this study, porcine GGTA1, CMAH, ß4GalNT2, vWF, ASGR1 genes were selected to introduce genetic modifications. The evaluation of three selected gRNAs within each gene was obtained, which enabled the selection of the best site for efficient introduction of changes. Modifications were examined after nucleofection of porcine primary kidney fibroblasts with CRISPR/Cas9 system genetic constructs, followed by the tracking of indels by decomposition (TIDE) analysis. In addition, off-target analysis was carried out for selected best gRNAs using the TIDE tool, which is new in the research conducted so far and shows the utility of this tool in these studies.