Bioassay Protocol Research Articles

Study on the cross-resistance of Aedes albopictus (Skuse) (Diptera: Culicidae) to deltamethrin and pyriproxyfen

BackgroundInsecticide resistance poses a significant challenge in the implementation of vector-borne disease control strategies. We have assessed the resistance levels of Aedes albopictus to deltamethrin and pyriproxyfen (PPF) in Fujian Province (China) and investigated the correlation between these resistance levels and mutations in the voltage-gated sodium channel (VGSC).MethodsThe WHO bioassay protocol was used to evaluate the resistance coefficient of Ae. albopictus to deltamethrin and PPF, comparing a susceptible population from the Foshan (FS) area with wild populations from the Sanming (SM), Quanzhou (QZ), Zhangzhou (ZZ), Putian (PT) and Fuzhou (FZ) areas in Fujian Province. Genomic DNA was analyzed by PCR and sequencing to detect knockdown resistance (kdr) in the VGSC, specifically at the pyrethroid resistance alleles V1016V, I1532I and F1534F. Molecular docking was also performed to analyze the binding interactions of PPF and its metabolite 4'-OH-PPF to cytochrome P450 (CYP) 2C19, 2C9 and 3A4 and Ae. albopictus methoprene-tolerant receptors (AeMet), respectively.ResultsThe analysis of resistance to deltamethrin and PPF among Ae. albopictus populations from the various regions revealed that except for the sensitive population in FS and the SM population, the remaining four regional populations demonstrated resistance levels ranging from 4.31- to 18.87-fold for deltamethrin and from 2.85– to 3.62-fold for PPF. Specifically, the FZ and PT populations exhibited high resistance to deltamethrin, whereas the ZZ and QZ populations approached moderate resistance levels. Also, the resistance of the FZ, PT and ZZ populations to PPF increased slowly but consistently with the increasing trend of deltamethrin resistance. Genomic analysis identified multiple non-synonymous mutations within the VGSC gene; the F1534S and F1534L mutations showed significant resistance to deltamethrin in Ae. albopictus. Molecular docking results revealed that PPF and its metabolite 4'-OH-PPF bind to the Ae. albopictus AeMet receptor and CYP2C19.ConclusionsThe wild Ae. albopictus populations of Fujian Province showed varying degrees of resistance to deltamethrin and PPF and a trend of cross-resistance to deltamethrin and PPF. Increased vigilance is needed for potential higher levels of cross-resistance, especially in the PT and FZ regions.Graphical

Exploring sources of inaccuracy and irreproducibility in the CDC bottle bioassay through direct insecticide quantification

BackgroundThe Centers for Disease Control and Prevention (CDC) bottle bioassay is a commonly used susceptibility test for measuring insect response to insecticide exposure. However, inconsistencies and high variability in insect response when conducting CDC bottle bioassays have been reported in previous publications. We hypothesized that the CDC bottle bioassay results may be compromised when expected and actual insecticide concentrations in the bottles are not equivalent and that inadequate bottle cleaning and/or loss during insecticide introduction and bottle storage steps could be responsible. We explored this hypothesis by quantifying insecticides using gas chromatography tandem mass spectrometry (GC-MS/MS) in bottles that had been cleaned, prepared, and stored according to the CDC guidelines.MethodsWe investigated the bottle cleaning, preparation, and storage methods outlined in the CDC bottle bioassay procedure to identify sources of irreproducibility. We also investigated the effectiveness of cleaning bottles by autoclaving because this method is commonly used in insecticide assessment laboratories. The two insecticides used in this study were chlorpyrifos and lambda-cyhalothrin (λ-cyhalothrin). Insecticides were removed from glass bioassay bottles by rinsing with ethyl-acetate and n-hexane and then quantified using GC-MS/MS.ResultsThe CDC bottle bioassay cleaning methods did not sufficiently remove both insecticides from the glass bottles. The cleaning methods removed chlorpyrifos, which has higher water solubility, more effectively than λ-cyhalothrin. Chlorpyrifos experienced significant loss during the bottle-coating process whereas λ-cyhalothrin did not. As for bottle storage, no significant decreases in insecticide concentrations were observed for 6 h following the initial drying period for either insecticide.ConclusionsThe CDC bottle bioassay protocol is susceptible to producing inaccurate results since its recommended bottle cleaning method is not sufficient and semi-volatile insecticides can volatilize from the bottle during the coating process. This can lead to the CDC bottle bioassay producing erroneous LC50 values. High levels of random variation were also observed in our experiments, as others have previously reported. We have outlined several steps that CDC bottle bioassay users could consider that would lead to improved accuracy and reproducibility when acquiring toxicity data.Graphical

The isolation of bioactive compounds from Warburgia ugandensis bark: A report of albicanyl acetate, caseamemin and β-sitosterol from Warburgia species

Warburgia ugandensis, which is one of the indigenous species of Ethiopia, is known for its wide range of biological activities. A series of drimane sesquiterpenoids have been isolated from the stem bark of the plant. However, there is no report on the herbicidal potential of the plant against invasive weeds like Parthenium hysterophorus. In this study, the herbicidal potential of W. ugandensis against the P. hysterophorus weed was investigated. Following the bioassay protocol, muzigadial as powerful phytotoxic compound together with other eight compounds were isolated from the EtOAc soluble portion of the ethanol extract of the bark of the plant. These compounds were identified using different physical and spectroscopic methods. The isolated compounds are albicanyl acetate (35), caseamemin (36), β-sitosterol (37), muzigadial (38), cinnamolide-3β-acetate (39), ugandensidial (40), 11α-hydroxy muzigadiolide (41), polygodial (42) and 9-deoxymuzigadial (43). The first three compounds are new to the species W. ugandensis. Furthermore, two other compounds namely heptacosanol (44) and hentriacontane (45) were also isolated from this species. In summary, the purpose of this study, to the best of my knowledge, is to provide the three initially identified compounds from the plant material and provide information on the plant's potential utility in agricultural applications.

Monitoring Aedes populations for arboviruses, Wolbachia, insecticide resistance and its mechanisms in various agroecosystems in Benin

Aedes mosquitoes are the main vectors of arboviruses in Benin. Cases of dengue have been reported in Benin with all four serotypes of the virus actively circulating in this region. Some agricultural settings are known to harbor Aedes vectors responsible for the transmission of arboviruses. The massive use of certain insecticides in agricultural settings has probably contributed to insecticide resistance in these vectors. In Benin, the susceptibility of arbovirus vectors to insecticides is poorly studied. In addition, the distribution of Wolbachia spp., which is used against some arboviruses is unknown. Moreover, there is limited information regarding the vectors responsible for the transmission of arboviruses in Benin. This present study monitored the species composition, arboviruses, and Wolbachia symbiont status, as well as the phenotypic and molecular insecticide resistance profile of Aedes populations from three agroecosystems in Benin. Aedes species identification was performed morphologically and confirmed using qPCR. (RT)-qPCR assay was applied for monitoring the presence of DENV, CHIKV, ZIKV, and WNV pathogens as well as for naturally occurring Wolbachia symbionts. Insecticide resistance was assessed phenotypically, by permethrin (0.75%) exposure of Adults (F0) using World Health Organization (WHO) bioassay protocols, and at the molecular level, using TaqMan (RT)-qPCR assays for assessing knock-down resistance (kdr) mutations (F1534C, V1016G/I, and S989P) and the expression levels of eight detoxification genes (P450s from the CYP9 and CYP6 families, carboxylesterases and glutathione-S-transferases). Aedes aegypti (Ae. aegypti) mosquitoes were the most abundant (93.9%) in the three agroecosystems studied, followed by Aedes albopictus (Ae. albopictus) mosquitoes (6.1%). No arboviruses were detected in the study's mosquito populations. Naturally occurring Wolbachia symbionts were present in 7 pools out of 15 pools tested. This could influence the effectiveness of vector control strategies based on exogenously introduced Wolbachia, all present in the three agroecosystems. Full susceptibility to permethrin was observed in all tested populations of Ae. albopictus. On the contrary, Ae. aegypti were found to be resistant in all three agroecosystem sites except for banana plantation sites, where full susceptibility was observed. Molecular analysis revealed that individual target site resistance kdr mutations F1534C and V1016G/I were detected in most Ae. aegypti populations. Additionally, double mutant (F1534C + V1016G/I) mosquitoes were found in some populations, and in one case, triple mutant (F1534C + V1016G/I + S989P) mosquitoes were detected. Metabolic resistance, as reflected by overexpression of three P450 genes (CYP6BB2, CYP9J26, and CYP9J32), was also detected in Ae. aegypti mosquitoes. Our study provides information that could be used to strategize future vector control strategies and highlights the importance of continuing vector surveillance. Future studies should assess the effect of piperonyl butoxide (PBO) on metabolic resistance and identify the different strains of Wolbachia spp., to choose the best vector control strategies in Benin.

Synthesis and efficacy of cinnamon oil formulations and their sustainable release against common house mosquito larvae

BackgroundControl of mosquitoes is considered an essential public health priority. This study was designed to estimate the larvicidal activity of two formulations of Cinnamomum zeylanicum EO for controlling Culex pipiens larvae.ResultsThe prepared formulations were a nanoemulsion of cinnamon (CNE), cinnamon (CN) alone and ordinary cinnamon essential oil mixed with sesame oil (CSO). The cinnamon + sesame oil (CSO) was added as one part cinnamon to 3 parts SO. Different concentrations were prepared and applied following the WHO larvicidal bioassay protocol. Our findings revealed that the LC50 of the CNE form ranged from 85.3 µg/mL to 28.30 µg/mL. The LC50 of SO alone was 1265 µg/mL but when mixed with CNE to form the CSO mixture, this decreased to 159.00 µg/mL. In terms of residual effect, the ordinary form of cinnamon had a residual effect in water for 72 h at a dose of 1000 µg/ml, but this extended to 120 h at the same dose when the CNE form was used. However CSO did not have a residual effect, however.ConclusionThe nanoemulsion form significantly improved the efficacy and residual effect of cinnamon against Culex pipiens larvae. Additionally, mixing cinnamon with sesame oil had a synergistic effect. This may assist control strategies against the house mosquito, Culex pipiens.

Ecotoxicology At School: A Proposal For Insertion Into Practical Science Classes

The complexity of environmental effects, caused by the diversity of chemical compounds interacting with the biota, brings to light the urgency of an emancipating education focused on integral human formation. In this sense, there is a concern about appropriately working with themes relevant to environmental education for students in the early years of primary education. Thus, educators in the field of biological sciences are allowed to approach topics related to biodiversity, to educate for citizenship, in the perspective that students recognize themselves as part of the environment. Problem-Based Learning - PBL involves questioning based on the observation of phenomena, hypothesis testing, and analysis of results, being an ally of science, considering that the student is the protagonist in the different stages of knowledge construction. Based on this methodology, auxiliary materials were prepared for use in the classroom, guided by concepts and playfulness. To contextualize the phenomena involved in the study of ecotoxicology, a survey was carried out of the contents and abilities of the Brazilian National Common Core Curriculum (BNCC, as per the acronym in Portuguese), listing the potentials to be examined together with the concepts of ecotoxicology. For these, bioassay protocols were chosen for the elaboration of practical activities that exemplify the thought phenomenon. Two skills were selected for the elaboration of stories that describe problematization, the accomplishment of the experimental activity, and the presentation of the results of the experiments throughout the character’s daily life. The area’s theoretical and practical framework involves using technical terms, which can compromise students’ interest and learning. To remedy the difficulty with the concepts, a mini dictionary was created with definitions and optimization of technical terms to simplify the research process and the student’s understanding.

Rapid-screening Bioassay Assessing Potential Allelopathic Influence on Spinach by Aqueous Extract from Fresh, Whole-plant Sorghum-sudangrass Tissue

Cover crops are defined as crops grown primarily for agroecosystem improvement rather than for market or sale. The use of cover crops to decrease the negative effects of weeds and improve soil and ecosystem health is increasing, but unanticipated allelopathic responses to those cover crops by subsequent crops is sometimes a problem. Allelopathy is broadly defined as the biochemical interactions between all types of plants, including microorganisms. Because allelopathic effects include both inhibitory and stimulatory responses and may be species- and cultivar-specific, a method of rapidly screening donor and recipient crops for allelopathic interactions is needed. The objective of this research was to evaluate a growth chamber bioassay for rapidly screening spinach (Spinacia oleracea L.) cultivars for allelopathic interactions with an aqueous extract from fresh whole-plant tissue of sorghum-sudangrass [Sorghum bicolor (L.) Moench; SSG] cultivars. The bioassay exposed the seed of 10 spinach cultivars to the aqueous extract of three cultivars of SSG during the imbibition and germination processes and evaluated the consequent root and stem development. Compared with the control, the extract from all three SSG cultivars decreased the root length of all spinach cultivars. A subsequent field screen where spinach cultivars were planted into decomposing SSG residue resulted in a similar pattern of growth suppression. These results demonstrate that the growth chamber bioassay is suitable for predicting allelopathic interactions between cultivars of SSG and cultivars of spinach and can be used by growers for making cultivar selection decisions when spinach follows SSG in a cropping sequence. This rapid-screening growth chamber bioassay protocol eliminates many of the environmental and other challenges frequently associated with field trials and may be adaptable for predicting allelopathic interactions among other cover crops, weeds, and subsequent market crops.

Distance-Regulated Photoelectrochemical Sensor "Signal-On" and "Signal-Off" Transitions for the Multiplexed Detection of Viruses Exposed in the Aquatic Environment.

Photochemical (PEC) sensors were severely limited for multiplex detection applications due to the cross interference between multiplex signals at the single recognition interface. In this work, a distance-regulated PEC sensor was developed for multiplex detection by using an i-Motif sequence with conformational transformation activity as the signal transduction unit. Through dynamic regulation of the spatial distance between the end site of the functional sequence and the electrode material, the photogenerated electrons on the surface of the sensor were directionally transferred. Thus, a PEC sensor with "signal-on" and "signal-off" dual signal output modes was developed for simultaneous detection of multitarget molecules. Combining isothermal nucleic acid amplification, the PEC sensor constructed in this work was successfully applied to the detection of two virus (Norovirus and Rotavirus) nucleic acid sequences. Under the optimal condition, this bioassay protocol exhibits a linear range of 0.01-100 nM for both viruses with detection limits of 0.72 and 0.53 pM, respectively. In this study, a stimulus-mediated distance regulation strategy successfully addressed the transduction of multiplex detection signals at the single recognition interface of the PEC sensor. It is expected that the technical barriers to multiplex detection of PEC sensors will be overcome and the application of PEC sensing technology will be expanded in the field of environmental analysis.

Bioassay Method Development to Test Sitophilus oryzae Sensitivity against Phosphine

Sitophilus oryzae is a cereal product pest found in warehouses. The presence of this pest negatively impacts the quality and quantity of stored rice. The common method used to control warehouse pests is fumigation. However, frequent use of insecticides will trigger resistance within target pest populations. This study aimed to develop a bioassay method based on the Food Agriculture Organization (FAO) protocols and modify bioassay protocols to determine S. oryzae susceptibility of populations collected from traditional markets across Yogyakarta. Field populations were collected from eight traditional markets in Yogyakarta (Lempuyangan Market [Kapanewon Danurejan], Kranggan Market [Kapanewon Jetis], Caturtunggal Market [Kapanewon Depok], Godean Market [Kapanewon Godean], Imogiri Market [Kapanewon Imogiri], Pleret Market [Kapanewon Pleret], Kasihan Market [Kapanewon Lendah], and Brosot Market [Kapanewon Galur]). The reference population was obtained from the Southeast Asian Regional Centre for Tropical Biology(SEAMEO BIOTROP). The bioassay was carried out following FAO protocols by using a glass jar fumigation chamber (volume 2 L) which was a modification of the desiccator of FAO recommended method number 16. This test used the lowest testable dose for this method: 0.01 × 10-2 mg/2L or 0.5 × 10-2 µg/L tested on 100 imagoes which were divided into five replications and obtained 100% mortality on the second day (48 hours). Results showed that the FAO fumigation chamber method could not be used in the test, so modifications were carried out to determine tested doses by changing the volume of the fumigation container. The test used three container volumes, including 20, 60, and 80 L. Result from 20 L container showed 82-100% mortality, 60 L containers showed 69-100% mortality, and 80 L containers showed 24-79% mortality. Results from 80 L container was suitable for the bioassay because it was able to test the lowest dose on reference insects. Eight doses ranging from 0.125 to 1.625 × 10-2 µg/L, and an untreated control were used to determine the LD50 of phosphine on each population. Insects were fumigated for 48 hours and then removed to observe mortality. The LD50 of the reference population was 0.27 × 10-2 µg/L while 0.29-0.54 × 10-2 µg/L for field populations. These findings indicate that S. oryzae populations collected from traditional markets in Yogyakarta were still susceptible to phosphine.

Biomarkers activity in Oreochromis niloticus under sub-chronic exposure to a UV filters ternary mixture

The behavior of organic UV filters in aquatic ecosystems and living organisms raises concern. For the first time, biochemical biomarkers were evaluated in the liver and brain of juvenile Oreochromis niloticus exposed to 0.001 and 0.5 mg L−1 of a benzophenone-3 (BP-3), octyl methoxycinnamate (EHMC), and octocrylene (OC) mixture for 29 days. Before the exposure, the stability of these UV filters was investigated using liquid chromatography. The experiment with aeration in the aquarium showed a high percentage of concentration reduction (%) after 24 h: 62 ± 2 for BP-3, 96 ± 6 for EHMC, and 88 ± 2 for OC versus 5 ± 4 for BP-3, 8 ± 7 for EHMC, and 2 ± 3 for OC when without aeration. These results defined the bioassay protocol. The stability of the filters concentrations after being stored in PET flasks and subjected to freezing and thawing cycles was also verified. In PET bottles, the BP-3, EHMC, and OC presented concentration reductions of 8 ± 1, 28 ± 7 and 25 ± 5 respectively, after 96 h storage and four freezing cycles. In falcon tubes the concentration reductions observed were 47 ± 2 for BP-3, >95 ± 1 for EHMC and 86 ± 2 for OC after 48 h and two cycles. The 29 days of sub-chronic exposure indicated the occurrence of oxidative stress through the enhanced lipid peroxidation (LPO) levels for the groups exposed to both bioassay concentrations. The catalase (CAT), glutathione-S-transferase (GST), and acetylcholinesterase (AChE) activities did not show significant alterations. The genetic adverse effects were analyzed in erythrocytes of fish exposed to 0.001 mg L−1 of the mixture by comet and micronucleus biomarkers and no significant damage was observed.

Visual/Photoelectrochemical Off-On Sensor Based on Cu/Mn Double-Doped CeO2 and Branched Sheet Embedded Cu2O/CuO Nanocubes

An integrated dual-signal bioassay was devised to fulfil thrombin (TB) ultrasensitive detection by integrating visualization with the photoelectrochemical technique based on G-quadruplex/hemin. During the process, branched sheet embedded copper-based oxides prepared with illumination and alkaline condition play a vital role in obtaining the desirable photocurrent. The switchover of photoelectrochemical signal was realized by the adjustable distance between electron acceptor G-quadruplex/hemin and interface materials due to dissociation of the Cu/Mn double-doped cerium dioxide (CuMn@CeO2)/DNA caused by the addition of TB. Then, CuMn@CeO2 transferred onto visual zones triggered catalytic reactions under the existence of 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide, making a variation in color recognized by the naked eye and providing visual prediction. Under optimized conditions, this bioassay protocol demonstrated wide linear ranges (0.0001-50 nM), high selectivity, stability, and reproducibility. More importantly, the proposed visual/photoelectrochemical transduction mechanism platform exhibits a lower background signal and more reliable detection results, which also offers an effective way for detecting other proteins and nucleic acids.

Effect of food resource and carton nest material on laboratory group survival of Nasutitermes corniger (Motschulsky, 1855) (Termitidae: Nasutitermitinae)

Ecological, behavioral, and toxicological studies with termites are frequently difficult to conduct under field conditions because their cryptic lifestyle. It is important to know the factors that can limit survival to ensure that laboratory studies will reflect the natural survival of these insects. Here, we tested the effect of a food resource and carton nest material on group survival of Nasutitermes corniger (Motschulsky, 1855) (Termitidae: Nasutitermitinae) in the laboratory. In general, termites survived longer in treatments with a food resource (sugarcane baits) compared to groups with carton nest material and control. However, groups with food resources exhibited high mortality due to fungal infection. The result of this study may contribute to the establishment of bioassay protocols performed with N. corniger in the laboratory.

SARS-CoV-2 spike protein detection using slightly tapered no-core fiber-based optical transducer.

The label-free detection of SARS-CoV-2 spike protein is demonstrated by using slightly tapered no-core fiber (ST-NCF) functionalized with ACE2. In the fabricated sensor head, abrupt changes in the mode-field diameter at the interfaces between single-mode fiber and no-core fiber excite multi-guided modes and facilitate multi-mode interference (MMI). Its slightly tapered region causes the MMI to be more sensitive to the refractive index (RI) modulation of the surrounding medium. The transmission minimum of the MMI spectrum was selected as a sensor indicator. The sensor surface was functionalized with ACE2 bioreceptors through the pretreatment process. The ACE2-immobilized ST-NCF sensor head was exposed to the samples of SARS-CoV-2 spike protein with concentrations ranging from 1 to 104 ng/mL. With increasing sample concentration, we observed that the indicator dip moved towards a longer wavelength region. The observed spectral shifts are attributed to localized RI modulations at the sensor surface, which are induced by selective bioaffinity binding between ACE2 and SARS-CoV-2 spike protein. Also, we confirmed the capability of the sensor head as an effective and simple optical probe for detecting antigen protein samples by applying saliva solution used as a measurement buffer. Moreover, we compared its detection sensitivity to SARS-CoV-2 and MERS-CoV spike protein to examine its cross-reactivity. In particular, we proved the reproducibility of the bioassay protocol adopted here by employing the ST-NCF sensor head reconstructed with ACE2. Our ST-NCF transducer is expected to be beneficially utilized as a low-cost and portable biosensing platform for the rapid detection of SARS-CoV-2 spike protein.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00604-022-05413-3.

Development and validation of a larval bioassay and selection protocol for insecticide resistance in Drosophila suzukii.

The rapid invasion of Drosophila suzukii (Matsumura) throughout Europe and the Americas has led to an increased reliance on calendar-based broad-spectrum insecticide programs among berry and cherry growers. Relatively few active ingredients (AIs) are currently available for effective D. suzukii management, and studies from multiple growing regions indicate that susceptibility to at least some of these materials is declining. Greater effort is needed to understand the status of susceptibility across field populations and the potential for increased resistance to develop, as well as the possible fitness costs incurred by resistant individuals. However, current bioassay protocols used for resistance monitoring and selection studies (i.e. resistance risk assessments) are labor-intensive and costly, making large-scale studies difficult to conduct. Here, we first present a novel bioassay protocol using larvae that requires little effort or cost to implement beyond what is needed for basic D. suzukii laboratory colony maintenance. We then perform dose-response bioassays using this protocol to identify larval lethal concentrations for three commonly used insecticides (malathion, spinosad and zeta-cypermethrin) in a susceptible population. Finally, resistance risk assessments were conducted using a population of D. suzukii from commercial caneberry fields near Watsonville, CA. We find that five generations of larval selection with a discriminating dose is sufficient to significantly increase both larval (malathion and spinosad) and adult (spinosad) resistance to the target AIs. This approach provides a simple, cost-effective tool for assaying susceptibility of D. suzukii populations to insecticides and for selecting resistant insect lines for resistance management research.

In vitro and in silico study of mixtures cytotoxicity of metal oxide nanoparticles to Escherichia coli: a mechanistic approach

Metal oxide nanoparticles (MONPs) are commonly found in the aquatic and terrestrial systems as chemical mixtures. Assessment of cytotoxicity associated with single and combination of MONPs can truly identify the concerned environmental risk. Thus, using Escherichia coli as a test model, in vitro cytotoxicity of 6 single MONPs, 15 binary and 20 tertiary mixtures with equitoxic ratios was evaluated following standard bioassay protocols. Assessment of oxidative stress suggested that the production of reactive oxygen species (ROS) was negligible, and the release of metal zinc ions played an important role in the toxicity of MONP mixtures. From our experimental data points, seven quantitative structure-activity relationships (QSARs) models were developed to model the cytotoxicity of these MONPs, based on our created periodic table-based descriptors and experimentally analyzed Zeta-potential. Two strategic approaches i.e. pharmacological and mathematical hypotheses were considered to identify the mixture descriptors pool for modeling purposes. The stringent validation criteria suggested that the model (Model M4) developed with mixture descriptors generated by square-root mole contribution outperformed the other six models considering validation criteria. While considering the pharmacological approach, the ‘independent action’ generated descriptor pool offered the best model (Model M2), which firmly confirmed that each MONP in the mixture acts through ‘independent action’ to induce cytotoxicity to E. coli instead of fostering an additive, antagonistic or synergistic effect among MONPs. The total metal electronegativity in a specific metal oxide relative to the number of oxygen atoms and metal valence was associated with a positive contribution to cytotoxicity. At the same time, the core count, which gives a measure of molecular bulk and Zeta potential, had a negative contribution to cytotoxicity.

Relationship between temperature and Anopheles gambiae sensu lato mosquitoes' susceptibility to pyrethroids and expression of metabolic enzymes

BackgroundMalaria remains one of the most devastating diseases globally, and the control of mosquitoes as the vector is mainly dependent on chemical insecticides. Elevated temperatures associated with future warmer climates could affect mosquitoes' metabolic enzyme expression and increase insecticide resistance, making vector control difficult. Understanding how mosquito rearing temperatures influence their susceptibility to insecticide and expression of metabolic enzymes could aid in the development of novel tools and strategies to control mosquitoes in a future warmer climate. This study evaluated the effects of temperature on the susceptibility of Anopheles gambiae sensu lato (s.l.) mosquitoes to pyrethroids and their expression of metabolic enzymes.MethodsAnopheles gambiae s.l. eggs obtained from laboratory-established colonies were reared under eight temperature regimes (25, 28, 30, 32, 34, 36, 38, and 40 °C). Upon adult emergence, 3- to 5-day-old female non-blood-fed mosquitoes were used for susceptibility tests following the World Health Organization (WHO) bioassay protocol. Batches of 20–25 mosquitoes from each temperature regime (25–34 °C) were exposed to two pyrethroid insecticides (0.75% permethrin and 0.05% deltamethrin). In addition, the levels of four metabolic enzymes (α-esterase, β-esterase, glutathione S-transferase [GST], and mixed-function oxidase [MFO]) were examined in mosquitoes that were not exposed and those that were exposed to pyrethroids.ResultsMortality in An. gambiae s.l. mosquitoes exposed to deltamethrin and permethrin decreased at temperatures above 28 °C. In addition, mosquitoes reared at higher temperatures were more resistant and had more elevated enzyme levels than those raised at low temperatures. Overall, mosquitoes that survived after being exposed to pyrethroids had higher levels of metabolic enzymes than those that were not exposed to pyrethroids.ConclusionsThis study provides evidence that elevated temperatures decreased An. gambiae s.l. mosquitoes' susceptibility to pyrethroids and increased the expression of metabolic enzymes. This evidence suggests that elevated temperatures projected in a future warmer climate could increase mosquitoes' resistance to insecticides and complicate malaria vector control measures. This study therefore provides vital information, and suggests useful areas of future research, on the effects of temperature variability on mosquitoes that could guide vector control measures in a future warmer climate.Graphical

Assessing the susceptibility to permethrin and deltamethrin of two laboratory strains of Phlebotomus perniciosus from Madrid region, Spain

Leishmania infantum is a protozoan causing cutaneous and visceral leishmaniasis in several regions of the world, including the Mediterranean basin. Phlebotomus perniciosus is one of the most important vectors of leishmaniasis in the countries of the western Mediterranean basin. Sand fly vector control by insecticides remains a useful tool in the framework of leishmaniasis control programs. Pyrethroids are the most widely used class of insecticides for sand fly control. There is currently a lack of information on the insecticide susceptibility and discriminating concentrations (DCs) of P. perniciosus. The aim of this study was to determine lethal concentrations (LC50, LC95, and LC99) and DCs of deltamethrin and permethrin against two strains of P. perniciosus from Madrid region (Spain). According to WHO tube bioassay protocol 24-h mortality obtained after 1-h exposure to deltamethrin (0.0003%, 0.001%, 0.003%, 0.01%, 0.03%, and 0.1%) and permethrin (0.003%, 0.01%, 0.03%, 0.1%, 0.3%, and 1%) was recorded. The LC50, LC95, and LC99 as well as their respective 95% confidence intervals values were calculated from the baseline data using maximum probability estimates of parameters and binary logistic regression analysis (QCal software). The 100% mortality was recorded from 0.01% of deltamethrin for both P. perniciosus strains and from 0.1% and 0.3% permethrin for Fuenlabrada and Boadilla strains, respectively. Final DCs of deltamethrin and permethrin of each P. perniciosus strain were determined based on setting this parameter at twice the minimum concentration of insecticide that kills 99% (LC99) at the following percentages: Fuenlabrada strain (0.0582% deltamethrin and 0.2648% permethrin) and Boadilla strain (0.0406% deltamethrin and 0.2446% permethrin). The results indicate that both P. perniciosus strains are susceptible to deltamethrin and permethrin and can be used in susceptibility tests, although Boadilla strain offers more consistent results. Due to the scarce existing literature on insecticide DCs for sand flies and the different current procedures to determine their susceptibility to insecticides it is a priority to multiply efforts in order to develop standards for monitoring insecticide resistance in sand flies.

Weight Gain of Spodoptera frugiperda Larvae (Lepidoptera: Noctuidae) on Leaf and Floral Tissues of Silphium integrifolium (Asterales: Asteraceae) Differs by Plant Genotype.

Silflower (Silphium integrifolium (Michaux)) is a native North American relative of sunflower that is undergoing domestication as a perennial oilseed crop. As silflower incurs pest damage from multiple insect species, it is necessary to screen genotypes for their effect on insect performance such that more pest tolerant/resistant accessions can be incorporated into future silflower breeding programs. We present a bioassay protocol for silflower using the generalist herbivore fall armyworm (Spodoptera frugiperda (J. E. Smith)). In this study, fall armyworm larvae were placed on leaf and flower tissue from eleven silflower genotypes, one cup plant (Silphium perfoliatum (L.) (Asterales: Asteraceae)) genotype, and an inbred sunflower line (Helianthus annuus (L.) (Asterales: Asteraceae), HA89). Caterpillar weight gained during a 4-d feeding period significantly differed on leaf and floral tissue from different silflower genotypes, between the Silphium species (silflower and cup plant), and between Silphium genotypes and annual sunflower. Two wild silflower genotypes produced lower larval weight gain on both the floral and leaf tissue than all other genotypes, suggesting these genotypes have either lower nutrition or greater resistance to fall armyworm. However, nonsignificant correlations between larval growth on floral versus leaf tissue across all plant species tested and among all silflower accessions suggest insect performances on these tissue types in silflower are independent. Along with identifying germplasm of interest for silflower breeding programs, we established an easily replicable bioassay protocol using fall armyworm on silflower floral and leaf tissues.

Effective residual life of bendiocarb insecticide on sprayed surfaces

Effective residual life of insecticides on sprayed surfaces determines the number of spraying cycles required to cover malaria transmission season. However, residual efficacy of insecticides varies with spray surface type. This study assessed effective residual life of bendiocarb on cow dung plastered and mud plastered wall surface types of human dwellings against An. arabiensis. Wall cone bioassays were carried out following the WHO (World Health Organization) standard operating procedures. Percentage mortality was calculated according to WHO bioassay protocol and Poisson regression model was fitted to show the association of mosquito mortality with explanatory variables using SAS (SAS Institute Inc. 2016. SAS® 9.4 Language Reference: Concepts, Sixth Edition. Cary, NC: SAS Institute Inc.). Efficacy of bendiocarb against An. arabiensis was 100% for the first 35 days on both cow dung and mud wall surface types. However, its efficacy deteriorated earlier on non-plastered wall surfaces after 35 days. The relationship between mosquito mortality and explanatory variables was: Mortality =3.637542, Sprayed = + 0.282841, Plastering= - 0.0094612 35-Days after spray, - 0.600159 65-Days after spray. Because coefficients for cone position were not significant, they were excluded from the regression equation. Generally, the effective residual life of bendiocarb on mud plastered wall surface was too short to cover malaria transmission season. Therefore, alternative insecticide with longer residual effective life is required in areas having more than two months of malaria transmission season.

Iron oxide nanoparticles exert inhibitory effects on N-Bis(2-hydroxypropyl)nitrosamine (DHPN)-induced lung tumorigenesis in rats

Iron oxide nanoparticles (magnetite) have been widely used in industry and medicine. However, the safety assessment of magnetite has not been fully completed. The present study was conducted to assess effects of magnetite on carcinogenic activity, using a medium-term bioassay protocol. A total of 100 male Fischer 344 rats, 6 weeks old, were randomly divided into 5 groups of 20 animals each, and given a basal diet and drinking water containing 0 or 0.1% of N-bis(2-hydroxypropyl)nitrosamine (DHPN) for 2 weeks. Two weeks later, the rats were intratracheally instilled magnetite 7 times at an interval of 4 weeks, at the doses of 0, 1.0 or 5.0 mg/kg body weight, and sacrificed at the end of the experimental period of 30 weeks. The multiplicities of macroscopic lung nodules and histopathologically diagnosed bronchiolo-alveolar hyperplasia, induced by DHPN, were both significantly decreased by the high dose of magnetite. The expression of minichromosome maintenance (MCM) protein 7 in non-tumoral alveolar epithelial cells, and the number of CD163-positive macrophages in tumor nodules were both significantly reduced by magnetite. It is suggested that magnetite exerts inhibitory effects against DHPN-induced lung tumorigenesis, by the reduction of alveolar epithelial proliferation and the M2 polarization of tumor-associated macrophages.