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.

An Optimized/Scale Up-Ready Protocol for Extraction of Bacterially Produced dsRNA at Good Yield and Low Costs.

Double-stranded RNA (dsRNA) can trigger RNA interference (RNAi) and lead to directed silencing of specific genes. This natural defense mechanism and RNA-based products have been explored for their potential as a sustainable and ecofriendly alternative for pest control of species of agricultural importance and disease vectors. Yet, further research, development of new products and possible applications require a cost-efficient production of dsRNA. In vivo transcription of dsRNA in bacterial cells has been widely used as a versatile and inducible system for production of dsRNA combined with a purification step required to extract the dsRNA. Here, we optimized an acidic phenol-based protocol for extraction of bacterially produced dsRNA at low cost and good yield. In this protocol, bacterial cells are efficiently lysed, with no viable bacterial cells present in the downstream steps of the purification. Furthermore, we performed a comparative dsRNA quality and yield assessment of our optimized protocol and other protocols available in the literature and confirmed the cost-efficiency of our optimized protocol by comparing the cost of extraction and yields of each extraction method.

Population-based change-point detection for the identification of homozygosity islands.

MOTIVATION: This work is motivated by the problem of identifying homozygosity islands on the genome of individuals in a population. Our method directly tackles the issue of identification of the homozygosity islands at the population level, without the need of analysing single individuals and then combine the results, as is made nowadays in state-of-the-art approaches. RESULTS: We propose regularized offline change-point methods to detect changes in the parameters of a multidimensional distribution when we have several aligned, independent samples of fixed resolution. We present a penalized maximum likelihood approach that can be efficiently computed by a dynamic programming algorithm or approximated by a fast binary segmentation algorithm. Both estimators are shown to converge almost surely to the set of change-points without the need of specifying a priori the number of change-points. In simulation, we observed similar performances from the exact and greedy estimators. Moreover, we provide a new methodology for the selection of the regularization constant which has the advantage of being automatic, consistent, and less prone to subjective analysis. AVAILABILITY AND IMPLEMENTATION: The data used in the application are from the Human Genome Diversity Project (HGDP) and is publicly available. Algorithms were implemented using the R software R Core Team (R: A Language and Environment for Statistical Computing. Vienna (Austria): R Foundation for Statistical Computing, 2020.) in the R package blockcpd, found at https://github.com/Lucas-Prates/blockcpd.

Method Validation and Evaluation of Safrole Persistence in Cowpea Beans Using Headspace Solid-Phase Microextraction and Gas Chromatography.

Bioinsecticides are regarded as important alternatives for controlling agricultural pests. However, few studies have determined the persistence of these compounds in stored grains. This study aimed at optimizing and validating a fast and effective method for extraction and quantification of residues of safrole (the main component of Piper hispidinervum essential oil) in cowpea beans. It also sought to assess the persistence of this substance in the grains treated by contact and fumigation. The proposed method used headspace solid-phase microextraction (HS-SPME) and gas chromatography with a flame ionization detector (GC/FID). Factors such as temperature, extraction time and type of fiber were assessed to maximize the performance of the extraction technique. The performance of the method was appraised via the parameters selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, and accuracy. The LOD and LOQ of safrole were 0.0057 and 0.019 µg kg-1, respectively and the determination coefficient (R2) was >0.99. The relative recovery ranged from 99.26 to 104.85, with a coefficient of variation <15%. The validated method was applied to assess the persistence of safrole residue in grains, where concentrations ranged from 1.095 to 0.052 µg kg-1 (contact) and from 2.16 to 0.12 µg kg -1 (fumigation). The levels measured up from the fifth day represented less than 1% of the initial concentration, proving that safrole have low persistence in cowpea beans, thus being safe for bioinsecticide use. Thus, this work is relevant not only for the extraction method developed, but also for the possible use of a natural insecticide in pest management in stored grains.

The efficacy of washing strategies in the elimination of fungicide residues and the alterations on the quality of bell peppers.

Food safety problems caused by pesticide residues in vegetables have become a top issue to raise public concern. In this study, bell peppers were grown in an experimental field and sprayed with two systemic (azoxystrobin and difenoconazole) and one contact (chlorothalonil) fungicides. Ozone (ozonated water and water continuously bubble with ozone) or conventional domestic (washing with distilled water, detergent, acetic acid, sodium bicarbonate, and sodium hypochlorite solutions) procedures were investigated to identify the most effective way to remove fungicide residues in bell peppers. The residues in the fruits and the washing solutions were determined by solid-liquid extraction with a low-temperature partition (SLE/LTP) and liquid-liquid extraction with a low-temperature partition (LLE/LTP), respectively, and analyzed by gas chromatography. Water continuously bubbled with ozone a concentration of 3 mg L-1 was the most efficient treatment with removal of fungicides residues ranging from 67% to 87%. However, similar treatment at a lower concentration (1 mg L-1) did not only efficiently removed fungicide residues (between 53% and 75%) but also preserving the quality of the fruit along a storage time of 13 days. Among the conventional solutions, sodium bicarbonate at 5% showed good efficiency removing between 60% and 81% of the fungicide residues from bell peppers, affecting the color quality of the fruit. Overall, the most affected physicochemical parameters in bell peppers after the treatments were weight loss, color, and vitamin C content.

Supreme Court v. Necropolitics: The Chaotic Judicialization of COVID-19 in Brazil.

Worldwide, governments have reacted to the COVID-19 pandemic with emergency orders and policies restricting rights to movement, assembly, and education that have impacted daily lives and livelihoods in profound ways. But some leaders, such as President Jair Bolsonaro in Brazil, have resisted taking such steps, denying the seriousness of the pandemic and sabotaging local control measures, thereby compromising population health. Facing one of the world's highest rates of COVID-19 infections and deaths, multiple political actors in Brazil have resorted to judicialization to advance the right to health and other protections in the country. Responding to this litigation has provided the country's Supreme Court an opportunity to assertively confront and counter the executive's necropolitics. In this article, we probe the malleable form and the constitutional basis of the Supreme Court's decisions, assessing their impact on the separation of powers, on the protection of human rights (for example, on those of prisoners, indigenous peoples, and essential workers), and relative to the implementation of evidence-based interventions (for example, lockdowns and vaccination). While the court's actions open up a distinct legal-political field (sometimes called "supremocracy")-oscillating between progressive imperatives, neoliberal valuations, and conservative decisions-the capacity of the judiciary to significantly address systemic violence and to robustly advance human rights remains to be seen.

Optimal Extraction of Ocimum basilicum Essential Oil by Association of Ultrasound and Hydrodistillation and Its Potential as a Biopesticide against a Major Stored Grains Pest.

The essential oil of basil (Ocimum basilicum) has significant biological activity against insect pests and can be extracted through various techniques. This work aimed to optimize and validate the extraction process of the essential oil of O. basilicum submitted to different drying temperatures of the leaves and extracted by the combination of a Clevenger method and ultrasound. The biological activity of the extracted oil under different conditions was evaluated for potential control of Sitophilus zeamais. The extraction method was optimized according to the sonication time by ultrasound (0, 8, 19, 31 and 38 min) and hydrodistillation (20, 30, 45, 60 and 70 min) and drying temperature (20, 30, 45, 60 and 70 °C). The bioactivity of the essential oil was assessed against adults of S. zeamais and the effects of each variable and its interactions on the mortality of the insects were evaluated. The best yield of essential oil was obtained with the longest sonication and hydrodistillation times and the lowest drying temperature of leaves. Higher toxicity of the essential oil against S. zeamais was obtained by the use of ultrasound for its extraction. The identification and the relative percentage of the compounds of the essential oil were performed with a gas chromatograph coupled to a mass selective detector. The performance of the method was assessed by studying selectivity, linearity, limits of detection (LOD) and quantification (LOQ), precision and accuracy. The LOD and LOQ values for linalool were 2.19 and 6.62 µg mL-1 and for estragole 2.001 and 6.063 µg mL-1, respectively. The coefficients of determination (R2) were >0.99. The average recoveries ranged between 71 and 106%, with coefficient of variation ≤6.4%.

Use of ozone and detergent for removal of pesticides and improving storage quality of tomato.

The efficiencies of two conventional domestic procedures (immersion in pure water and detergent solution at 0.25 and 1%) and two treatments using ozone (immersion in water with bubbling O3 and immersion in ozonated water, both at 1 and 3 mg L-1) were evaluated for the removal of residues of the fungicides azoxystrobin, chlorothalonil and difenoconazole in tomatoes. The fungicides were sprayed on the fruits at the recommended concentration for the crop. The residues in the tomatoes and in the washing solutions were determined by extraction with low-temperature partition techniques and analysis by gas chromatography. More concentrated solutions were more effective in removing pesticide residues. The water bubbled with ozone at 3 mg L-1 was the most efficient treatment for the removal of fungicides, reaching a reduction of 70-90% of the residues. However, the treatments with the lowest concentration of ozone had lower loss of fruit mass during storage.

Eugenol diffusion coefficient and its potential to control Sitophilus zeamais in rice.

Given the insecticidal potential of eugenol as a fumigant, this work aimed to determine the diffusion coefficient of eugenol emanating from a pure standard solution (99%), as well as from clove essential oil (Eugenia caryophillata Thunb. (Myrtaceae)) through rice grain; to chemically analyse the volatile composition of commercially available eugenol and clove essential oil; and to evaluate the mortality of Sitophilus zeamais Motschulsky (Coleoptera: curculionidae) after exposure to eugenol inside a test chamber filled with rice. The solid phase microextraction method of extracting and quantifying eugenol by gas chromatography presented a good analytical response for the quantification of the analyte. There was no significant difference between the diffusion coefficient of eugenol diffusing from pure eugenol or from clove essential oil. The diffusion coefficient of eugenol through rice with the conditions herein adopted is 1.09 × 10-3 cm2 s-1. The characterization of clove essential oil confirmed the presence of eugenol as its major component (74.25%). A difference was observed in the composition of the distinct phases evaluated. The exposure of adult S. zeamais to diffused eugenol from pure eugenol over seven days resulted in significantly higher mortality rates (~37%) than eugenol diffused from clove essential oil (~11%). No differences in mortality rates were observed in individuals placed at different positions inside the test chamber during eugenol fumigation.

Insecticidal activity of Vanillosmopsis arborea essential oil and of its major constituent α-bisabolol against Callosobruchus maculatus (Coleoptera: Chrysomelidae).

Vigna unguiculata, one of the most important legumes, mainly in underdeveloped countries, is susceptible to post-harvest losses in storage by Callosobruchus maculatus (Fabricius, 1775) (Coleoptera: Chrysomelidae). The work evaluated the toxicity, inhibition of oviposition, instantaneous rate of population growth (ri) and the development of fumigated C. maculatus with the essential oil of Vanillosmopsis arborea and its major constituent, α-bisabolol. The experimental units consisted of 0.8 L flasks treated with concentrations of 1.2-11.2 µL L-1of air of the essential oil of V. arborea or its major constituent applied to disks of filter paper. α-Bisabolol was quantified as 409.33 mL L-1 of the essential oil. The development rate of C. maculatus was evaluated by daily adult counts. Oviposition was evaluated at lethal concentrations (LC50, LC25, LC10 and LC1). The LC50 and LC95 of the essential oil of V. arborea and α-bisabolol were 5.23 and 12.97 µL L-1 of air and 2.47 and 8.82 µL L-1 of air, respectively. At some concentrations, the α-bisabolol was more toxic to males than to females of the insect. Increased concentrations of the essential oil reduced the ri, rate of development, oviposition, and number of eggs of C. maculatus and therefore have potential for pest control.

Difenoconazole and linuron dissipation kinetics in carrots under open-field conditions.

The dissipation of difenoconazole and linuron using an open-field experimental approach with carrots exposed to one-, two- and fivefold the recommended dose of the pesticides was evaluated to provide safe recommendation to ensure food safety of carrots. The pesticide residue analysis was performed with solid-liquid extraction with low temperature partitioning technique (SLE/LTP) followed by gas chromatography analysis. The recovery percentages of extracts obtained from samples of carrot passed through SLE/LTP extraction and fortified with difenoconazole and linuron pesticides varied from 93.4% to 106.3% and from 95.1% to 116.6%, respectively. The limit of detection for difenoconazole was 0.02 and 0.12 mg kg-1 for linuron. The limit of quantification for difenoconazole was 0.05 and 0.36 mg kg-1 for linuron. The degradation time for fifty percent of the applied pesticide at the different doses ranged from 2.4 to 3.6 days for difenoconazole and from 7.5 to 10.5 days for linuron. At the end of the pre-harvest interval, carrots treated with fivefold the recommended dose of both pesticides were considered unfit for consumption. Despite monitoring the degradation products of the applied pesticides by gas chromatography coupled to mass spectrometer, none degradation product was identified on the carrots.

Ozone treatment for pesticide removal from carrots: Optimization by response surface methodology.

The present study aimed to optimize ozone (O3) treatments, as gas and dissolved in water, to remove difenoconazole and linuron in carrots. We employed a central composite design to study three variables governing the efficacy of treatments: O3 concentration, temperature and treatment time. The temperature did not influence the efficacy of treatments. The removal percentage of pesticides increases with increases in ozone concentration and the time of treatment. O3 application promoted the removal of more than 80% of pesticides when the roots were exposed for approximately 120min at 5 and 10mgL-1, respectively, in treatments with O3 as gas and dissolved in water. After storage, pesticide removal was higher than 98% for difenoconazole and 95% for linuron. The degradation products from the pesticides resulting from treatment were monitored, but none were found. This is the first report demonstrating the removal of difenoconazole and linuron from carrots by ozone.