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Clothianidin

Clothianidin is a neonicotinoid insecticide used to control a varity of pests on agricultural crops.
It acts as an agonist at the nicotinic acetylcholine receptor, disrupting nerve impulse transmission and leading to paralysis and death in target insects.
Clothianidin is commonly applied as a seed treatment or foliar spray, and has been shown to have toxic effects on non-target organisms like bees and other pollinators.
Researchers studying Clothianidin's mechanisms of action, environmental fate, and impacts on ecosystems can utilize the PubCompare.ai platform to optimize their research through AI-powered literature analysis and protocol comparison tools.
This can enhance the reproducibility and accuracy of Clothianidin studies, advancing our understadning of this important insecticide.

Most cited protocols related to «Clothianidin»

1
Environmental Toxicity Loading of Insecticides
Cited 6 times
Our approach provides a general measure of acute toxicity loading of insecticides on US agricultural land and surrounding areas, assuming insects are exposed to pesticides released to the environment through direct contact with contaminated surfaces, water, or food or through ingestion of contaminated food or water. Different insects will have different exposures depending on their habitat, behaviors, and food sources; however, across years, exposures for different types of insects will be comparable. However, as noted previously, this analysis does not include actual or estimated exposure doses, nor does it factor in timing and mode of pesticide application. Therefore, the AITL method would best be described as a screening analysis that can identify or predict potential environmental impacts.
Honey bee lethality is the measure of toxicity used to assess AITL. This analysis was developed for both contact toxicity (AITLc) and oral toxicity (AITLo). The AITLC calculation provides the number of toxicity loading units (TLU) applied to a crop for each pesticide by dividing the mass of chemical applied (in μg) by the honey bee contact LD50 (in μg/bee) (the first term in Eq 1 below) to give the number of honey bee LD50’s released to the environment. This value is then modified by the half-life of the chemical (in days), assuming exposure continues as long as the chemical is present, with degradation governed by the half-life of the chemical and the dose expressed as the area under the curve of concentration versus time (second term in Eq 1). Because the AITL values obtained are on the order of 1012–1018, a scaling factor of 10−15 is included to scale the values for plotting the results. The same method of calculation is applied for AITLo (Eq 2).
Toxic degradates are known for some pesticide active ingredients. However, because environmental half-lives were not available for most of these compounds they were not included in the analysis. Those degradates with known toxicity (e.g., malaoxon, the degradate of malathion) might contribute to overall acute toxicity, although we determined that most known degradates would contribute only a negligible amount to the overall toxicity loading of the parent compound. The one exception as noted previously is clothianidin, which is a metabolite of thiamethoxam; our analysis accounts for this conversion in the environment because it contributes a measurable level of toxicity relative to the parent compound.
We estimated pesticide loading on agricultural land and surrounding areas as the area under the curve of degradation/dissipation of pesticides over time, assuming typical first-order kinetics, as recommended by US EPA in its guidance [38 ]. While degradation rates vary depending on a number of factors, the first-order assumption is widely used for estimating pesticide concentrations in the environment over time, and this appears to be an appropriate assumption for the neonicotinoid insecticides [39 (link), 40 (link)]. An example theoretical degradation curve for imidacloprid, with a half-life of 174 days, is shown in Fig 3. In this example, on Day Zero (application day), the available dose is 150 honey bee LD50s. On Day One, 149 honey bee LD50s still remain, with the potential for concomitant toxic effects to insects. On Day 174, 75 honey bee LD50s remain in the environment. Ninety-seven percent of the imidacloprid is degraded at five half-lives (870 days or 2.4 years). The total integrated environmental toxicity loading level over time can be calculated as the area under the curve. Therefore, we define AITL as the area under the curve in number of honey bee LD50-days, representing the total exposure potential for arthropods (both terrestrial and aquatic) over the degradation period.
For pesticides used as seed treatments, our analysis assumes that insect exposure from contact with treated crops would include dust drift to field-side plants during seed planting (which can be considerable) resulting in both contact and oral exposure, and oral exposure from consuming pollen, nectar, guttation droplets, or plant tissue from the treated crop [12 (link)]. In addition, application of the seeds to soil would result in exposure of the soil entomofauna and migration to waterways would result in exposures for aquatic insects. This is a simplifying assumption, which may or may not overestimate actual insecticide doses received by honey bees and other beneficial insects from seed treatments, depending on the specific circumstances. Based on a “residue per unit dose” estimation, it appears that seeding results in higher contamination of insects than an equivalent spray application but, due to the lower per hectare (or acre) rates of application for seed treatments, a comparable level of contamination in non-target arthropods can be expected [41 ]. Because the AITL is intended to be used as a screening level assessment for comparative and surveillance purposes, the inclusion of seed treatment applications is a reasonable approach. Further refinement of this method or other analyses would be required before making policy or regulatory decisions based on seed insecticide treatments alone.
DiBartolomeis M., Kegley S., Mineau P., Radford R, & Klein K. (2019). An assessment of acute insecticide toxicity loading (AITL) of chemical pesticides used on agricultural land in the United States. PLoS ONE, 14(8), e0220029.
Publication 2019
2
Investigation of Bee Colony Collapse Disorder
Cited 5 times
During the spring of 2011, we again received reports of dead and dying bees at a local apiary, located in a small wooded area near maize and soybean production fields in northwestern Indiana. As in 2010, these reports coincided with local planting and tillage activities. We collected bees from the entrances of several hives on May 10th and 12th, 2011. We also collected apparently healthy returning foragers from hives at the same apiary. We removed frames containing nectar and pollen from two colonies at this location: one frame was taken from a hive with dead bees near its entrance, the second frame was removed from a nearby hive without any dead bees visible. Pollen and nectar from these frames was removed from cells for analysis, and two separate analyses of pollen samples were conducted. Finally, we collected samples of surface soil (using the methods outlined above) and dandelion flowers (multiple areas sampled, approximately 7–10 flowers were collected/sample) from maize fields within 2 km of this apiary that were being planted at the time of our bee collections.
We also collected additional waste talc samples in 2011, using commercially available neonicotinoid treated maize seed from several different manufacturers. Because our goal was to develop a representative sample from a variety of maize hybrids used in our research area, all hybrids were selected based upon agronomic suitability for local planting. Both clothianidin and thiamethoxam treated seed was used, at application rates ranging from 0.25 mg/kernel to 1.25 mg/kernel. Talc was added to each seed box at the recommended rate (approx. 240 cc talc/75 kg of maize seed) (36). Fields were planted using a 6-row John Deere 7200 MaxEmerge planter. Collection of waste talc for analysis was performed following planting by manually removing approximately 50 g of talc from the manifold of the planter vacuum system using a scoopula. The planter and vacuum system was exhausted thoroughly and cleaned with compressed air prior to each planting and following each collection.
Krupke C.H., Hunt G.J., Eitzer B.D., Andino G, & Given K. (2012). Multiple Routes of Pesticide Exposure for Honey Bees Living Near Agricultural Fields. PLoS ONE, 7(1), e29268.
Publication 2012
Cells clothianidin Flowers Hybrids Neonicotinoids Plant Nectar Pollen Reading Frames Soybeans Talc Taraxacum Thiamethoxam Urticaria Vacuum Zea mays
3
Initial IRS Impact on Mosquito Mortality
Cited 5 times
The first analysis summarises and compares the initial impact of different IRS products. Data were restricted to initial timepoints collected within 2 months of IRS application as the active ingredient decays with time, so that averaging across the whole dataset may mis-represent the initial potency of IRS as studies had different durations. Statistical models were fit to generate overall estimates of the efficacy of the chemical class. These explanatory factors included the mosquito vectors (classified at the species complex level and species level where possible, i.e. A. arabiensis, A. funestus s.l. and A. gambiae s.l.), experimental hut type (West or East African design) and hut wall substrate (cement or mud) alongside the chemical class used for the IRS (carbamate, clothianidin, organophosphate and pyrethroid). Preliminary data exploration revealed that there were too few data to perform an extensive statistical test on all covariates. To overcome this a subset of the full database was generated by removing Ifakara hut studies, wall substrates that were not mud or cement and chemistries other than pyrethroids, organophosphates, carbamates or neonicotinoids. Binomial logistic regression models were fitted to the remaining count data (N = 78) to estimate the number of mosquitoes that were dead in 24-h, had exited, blood-fed or been deterred by the IRS product. The predicted value for the proportion of mosquitoes being killed, exiting, blood-fed or deterred is calculated as: πi=logit-1lnπi∕1-πi=expβ0+∑hβhXhi1-expβ0+∑hβhXhi where πi is the estimated proportion for the ith data (e.g. the proportion of mosquitoes killed), β0 is the intercept, the subscript h denotes the covariate of interest (taking number of 1 to H) and Xh is a matrix of explanatory factors (mosquito species, hut type, substrate and chemistry sprayed) with coefficients βh59 (link). Bayesian models were fitted using Hamiltonian Monte Carlo sampling methods60 (link),61 . Four chains were initialised to assess the convergence of 2000 iterations, the first 1000 of each were discarded as burn in. The posterior distributions of parameters (4000 iterations) and 90% Bayesian credible intervals were estimated, posterior checks were performed using ShinyStan (version 1.0.0)62 and visually confirmed to fit the data (Supplementary Fig. 2–5).
Sherrard-Smith E., Griffin J.T., Winskill P., Corbel V., Pennetier C., Djénontin A., Moore S., Richardson J.H., Müller P., Edi C., Protopopoff N., Oxborough R., Agossa F., N’Guessan R., Rowland M, & Churcher T.S. (2018). Systematic review of indoor residual spray efficacy and effectiveness against Plasmodium falciparum in Africa. Nature Communications, 9, 4982.
Publication 2018
A-factor (Streptomyces) BLOOD Carbamates clothianidin Culicidae Dental Cementum East African People factor A IRS2 protein, human Mosquito Vectors Neonicotinoids Organophosphates Pyrethroids
4
Neonicotinoid Exposure Impacts on Bee Foraging
Cited 5 times
Experiments were performed at Trinity College, Dublin with Bombus terrestris dalmatinus (Unichem Ltd, Co. Dublin, Irish distributor for Koppert). Colonies were maintained at 25-30 °C in 24 h darkness and fed commercial pollen and Biogluc (Agralan Ltd, Swindon) bee food ad libitum. Experiments were also performed at Newcastle University, Newcastle upon Tyne with Bombus terrestris audax (Biobest, Belgium) and Bombus terrestris terrestris (Koppert Biological Systems, NATURPOL, Netherlands). Bees from 3-5 different colonies were used for each neonicotinoid. Individual worker bumblebees were collected as they tried to exit the colony. For the experiments with newly-emerged bumblebees, colonies were monitored for newly emerged bees daily; newly-emerged adults were identified by their pale colour. These bees were extracted using forceps from within the colony. As previously described in Tiedeken et al. (2014)16 (link), individual bumblebees were cold anesthetized, weighed and sex-determined, and transferred to individual 650 ml plastic containers (160×110×45mm). Containers were fitted with three 3 ml feeding tubes, inserted horizontally. Feeding tubes had four 2 mm holes so bees could alight on the tubes and feed from the openings. The feeding tubes contained one of three solutions: (1) deionized water; (1) 0.5M sucrose; or (3) 0.5M sucrose with a specific concentration of a neonicotinoid compound. Whether or not the bee was alive was noted 24 h after start of experiment. Bees that did not drink from either tube were excluded from the final analysis; the total number of these subjects was never greater than 3 per treatment (note: these subjects were always dead and likely to have died from stress or other causes).
Experiments with honeybees (Apis mellifera var Buckfast) were performed at Newcastle University during the summer months using 2 free-flying outdoor colonies originally obtained from the UK’s National Bee Unit (Sand Hutton, Yorkshire). Foraging adult worker honeybees were collected at the colony entrance as they returned from foraging; newly-emerged adult workers were collected from brood comb as they emerged in a purpose built box kept in an incubator at 34°C. Bees were cold anesthetized prior to placing in rearing boxes. Cohorts of 25 bees were placed in rearing boxes as previously described in Paoli et al. (2014)31 (link). Four food tubes (as described above) were provided: (1) one with deionized water; (2) two with 1M sucrose; (3) two with 1M sucrose containing a specific concentration of a neonicotinoid. The number of bees alive in each cohort was counted at the time of measurement of the food consumption (24 h later).
All of the two-choice experiments were performed experimenter-blind (except IMD with bumblebees). Three neonicotinoid pesticides, imidacloprid (IMD), thiamethoxam (TMX) and clothianidin (CLO), were used in the experiments (Pestanal®, Sigma-Aldrich). The neonicotinoid concentrations used were 1nM, 10nM, 100nM, 1μM (see Extended Data Table 2 for conversions to ppb and ng/bee). Bees were kept in continuous darkness for 24 h at constant temperature and 60% RH (bumblebees: 28 °C; honeybees: 34 °C). Control boxes identical to the experimental boxes (without bees) for each neonicotinoid treatment were placed in the incubator simultaneously with the experiments to measure the rate of evaporation from the food solutions. Feeding tubes were weighed, placed in the experimental boxes with the bees for 24 h, and then removed and weighed a second time. The position of the treatment tubes was randomized across subjects. The amount of solution consumed was determined as the difference in the weight of each tube after 24 h; the average value for the evaporation control for each treatment was subtracted from this final value for each tube. For bumblebees, sample sizes were: IMD: 1nM = 57, 10nM = 66, 100nM = 65, 1μM = 66; TMX: 1nM = 38, 10nM = 39, 100nM = 36, 1μM = 40; CLO: 1nM = 57, 10nM = 59, 100nM = 48, 1μM = 62. For honeybees, N = 40 cohorts of 25 bees/treatment. Sample size was chosen as N ≥ 40 based on previous work16 (link); sample size varied because some individuals died from unknown causes at the start of the experiments.
Kessler S., Tiedeken E.J., Simcock K.L., Derveau S., Mitchell J., Softley S., Stout J.C, & Wright G.A. (2015). Bees prefer foods containing neonicotinoid pesticides. Nature, 521(7550), 74-76.
Publication 2015
Adult Apis Audax Bees Biopharmaceuticals clothianidin Cold Temperature Comb Darkness Food Forceps imidacloprid Neonicotinoids Pesticides Pollen Sucrose Thiamethoxam Tube Feeding Visually Impaired Persons Workers
5
Antioxidant Effects of Catuaba on Stressed Mice
Cited 5 times
Experimental animals: Male C57BL/6NCrSlc mice (8 weeks old) were purchased
from Japan SLC (Hamamatsu, Japan). All mice were maintained in 40.5 × 20.5 × 18.5 cm
individual ventilated cages (Sealsafe Plus Mouse; Tecniplast, Buguggiate, Italy) under
controlled temperature (23 ± 2°C) and humidity (50 ± 10%) on a 12-hr light/dark cycle at the
Kobe University Life-Science Laboratory with ad libitum access to a pellet
diet (DC-8; Clea Japan, Tokyo, Japan) and filtered water. This study was approved by the
Institutional Animal Care and Use Committee (Permission #24-10-03) and carried out according
to the Kobe University Animal Experimental Regulation.
CTD purification and HPLC analysis: Water-soluble Dantotsu®(involving 16% of CTD; Sumitomo Chemical Co., Tokyo, Japan), donated by Sado City (Niigata,
Japan), was washed with 10 times the amount of distilled water to remove the surfactant
activating and granulating agents. After being left to stand for at least 48 hr, the
supernatant was removed. This step was repeated five times, and then, the white precipitate
was collected and air-dried naturally for a week. The content rate of CTD in the white
precipitate was measured by a LaChrom high-performance liquid chromatography (HPLC) system
(interface L-7000, pump L-7100, auto sampler L-7200, column oven L-7350 and UV-VIS detector
L-7420; Hitachi, Tokyo, Japan) using a Capcell Pak C18 UG120 column (5 µm
particles, 4.6 × 250 mm; Shiseido, Tokyo, Japan).
CTD standard (>99.8%; Wako Chemical, Osaka, Japan) and the obtained white powder were
completely dissolved in dimethyl sulfoxide (DMSO) and then serial-diluted with a mobile
phase consisting of 55% acetonitrile in 50 mM potassium phosphate buffer (pH 3.0), followed
by filtration with a 0.20 µm syringe driven filter unit (Millex-LG;
Millipore, Billerica, MA, U.S.A.). The column maintained at 40°C was eluted with the mobile
phase at a flow rate of 1.0 ml/min. After the column was equilibrated, 10
µl of the samples was injected into the HPLC system. We monitored the
resultant chromatograph at the wavelength of 260 nm and then calculated the CTD content of
the obtained white powder from the calibration curve created by the peak areas and heights
of the CTD standard. A single peak was observed in the samples from the white precipitate
with the same retention time as that of the CTD standard. A linear calibration curve
(R2=0.999) created from samples serial-diluted with the CTD standard showed
that our purification makes the content rate of CTD increased from 14–16% to 93–97% by
weight.
CTD administration and stress exposure: All mice were allowed to acclimate
to their home cages for a week prior to the initiation of experiments. We divided the mice
into eight groups (n=5 mice in each cage): CTD-0 (Control), CTD-10 (10 mg/kg/day), CTD-50
(50 mg/kg/day) and CTD-250 (250 mg/kg/day) with the presence or absence of stress exposure.
As a substitute for filtered water for the mice, we used the MediGel® Sucrarose 2
oz cup (ClearH2O, Portland, ME, U.S.A.), which is a flavored thermoreversible
hydration gel matrix. The amounts of the purified CTD for the respective administration
groups were calculated from the CTD purity (95%), daily gel intake (5 g/day/mouse), total
gel weight (60 g; excluding the package weight) and average mouse weight (24 g; weighed at
initiation of experiments). These amounts of CTD were completely dissolved in 600
µl DMSO (1% volume of a gel) and injected into the gels and then
double-boiled at 60°C followed by shaking to ensure that the CTD was diffused well. For the
CTD-0 group, the same volume of DMSO without the purified CTD was injected into the gels.
All gels were strapped with cable ties under the grate of the cage lid to prevent
contamination with the beddings and the excretions. All gels were weighed daily to estimate
the amounts of the putative CTD exposure. In the four stress groups, the mice were subjected
to an unpredictable chronic stress procedure as described in our earlier report [14 (link)] with some modifications. Briefly, the following six
stressors were used: 5 min forced swimming in water at room temperature (RT), 24 hr food and
water deprivation, continuous overnight illumination, 30 min horizontal cage shaking (80
rpm), 24 hr switching of cagemates (being housed with another mouse) and 24 hr wet bedding.
To maximize the unpredictability of this paradigm, the mice were randomly exposed to two
mild stressors per day at varying times for 4 weeks.
Behavioral analysis: On the last day of the 4 week experimental period, an
open field test was conducted during the light phase to evaluate the locomotor activity and
the anxiety-like behavior of the mice. Briefly, the mouse was placed on the corner of an
open field (60 × 60 × 30 cm) with LED illumination. All of the mouse’s activities were
recorded by a video camera for the subsequent 10 min, and we then analyzed the total
distance traveled and the time spent in the center zone (30 × 30 cm) using SMART video
tracking software V3.0 (San Diego Instruments, San Diego, CA, U.S.A.).
Tissue preparation: On the day after the completion of the 4 weeks of
combined exposure to CTD and stress, all mice were deeply anesthetized with diethyl ether
and transcardially perfused with 0.9% normal saline, followed by perfusion with ice-cold 4%
paraformaldehyde in phosphate buffer. The testes were excised, weighed and postfixed with
the same fixative overnight at 4°C. The testes were dehydrated through a graded series of
ethanol followed by xylene and embedded in paraffin. Serial sections of testes were then cut
at 4 µm thickness on a sliding microtome (SM2000R; Leica Microsystems,
Wetzlar, Germany) and mounted on slide glasses (Platinum Pro; Matsunami Glass Ind.,
Kishiwada, Japan). All sections were stored at −30°C until use for the following steps.
Histological and immunohistochemical analyses: For the general
histological analysis, testis sections were stained with hematoxylin and eosin (HE; Merck
KGaA, Darmstadt, Germany) after their deparaffinization and hydration, following the
manufacturer’s instructions. To detect antioxidant enzymes in the testes, we performed the
following immunohistochemistry protocol. The sections were immersed in absolute methanol and
0.5% H2O2 for 30 min, respectively, at RT to quench the endogenous
peroxidase activity. They were then incubated with Blocking OneHisto (Nacalai Tesque, Inc.,
Kyoto, Japan) for 1 hr at RT for protein blocking and then incubated with the rabbit
polyclonal anti-GPx4 antibody (Item No. 10005258; Cayman Chemicals, Ann Arbor, MI, U.S.A.)
diluted 1:8,000 in phosphate buffered saline with 0.05% Tween-20 (PBST; pH 7.4) for 18 hr at
4°C.
After being washed with PBST, the sections were reacted with goat anti-rabbit
immunoglobulins conjugated to peroxidase-labeled dextran polymer in tris (hydroxymethyl)
aminomethane-HCl buffer (EnVision+; Dako, Glostrup, Denmark) for 1 hr at RT.
Immunoreactivity was then detected by incubation with 3,3′-diaminobenzidine solution
(EnVision+ kit/HRP[DAB], Dako). The sections were then rinsed with distilled water and
counterstained lightly with hematoxylin solution for 1 min. Next, the sections were placed
in a graded series of ethanol, dehydrated with absolute ethanol, cleared by xylene and
coverslipped with Eukitt (O. Kindler GmbH, Freiburg, Germany).
Statistical analysis: Statistical analyses were performed with Excel
Statistics 2012 (SSRI version 1.00, Tokyo, Japan). In the behavioral analyses, outliers more
distant than 1.5 interquartile ranges from the upper or lower quartile were omitted. All
data were analyzed by two-way ANOVA (CTD ×stress) followed by the Tukey-Kramer’s post hoc
test. The results were considered significant when the P-value was less
than 0.05.
HIRANO T., YANAI S., OMOTEHARA T., HASHIMOTO R., UMEMURA Y., KUBOTA N., MINAMI K., NAGAHARA D., MATSUO E., AIHARA Y., SHINOHARA R., FURUYASHIKI T., MANTANI Y., YOKOYAMA T., KITAGAWA H, & HOSHI N. (2015). The combined effect of clothianidin and environmental stress on the behavioral and reproductive function in male mice. The Journal of Veterinary Medical Science, 77(10), 1207-1215.
Publication 2015

Most recents protocols related to «Clothianidin»

1
Clothianidin's Insecticidal Mechanism and Public Health Applications
Insects possess nicotinic acetylcholine receptors (nAChR), which are the target of clothianidin in their nervous system [43 (link)]. These receptors are responsible for the transmission of nerve signals between nerve cells. Clothianidin acts by binding specifically to these nAChR receptors. When it binds to nAChR receptors, it activates them for an extended duration compared to acetylcholine, which is a natural neurotransmitter. This results in excessive stimulation of nerve cells and prolonged excitation of the insect’s nervous system [44 (link)]. Consequently, the insect becomes paralyzed because its nervous system remains constantly excited and can no longer function properly. Eventually, this leads to the insect’s death [43 (link)–45 (link)].
Clothianidin 50 WG was introduced for public health use in Benin in 2021 as part of large-scale community-based indoor residual spraying (IRS) campaigns. This choice aimed to optimize IRS by reducing infectivity through the elimination of malaria vectors, particularly those with resistance mechanisms such as kdr-L995F and ace-1 G280S, with the new chemical mode of action of clothianidin.
Odjo E.M., Tognidro M., Govoetchan R., Missihoun A.A., Padonou G.G., Ahouandjinou J.M., Akinro B., Koukpo Z.C., Tokponnon F.T., Djenontin A., Agbangla C, & Akogbeto M.C. (2024). Malaria transmission potential of Anopheles gambiae s.l. in indoor residual spraying areas with clothianidin 50 WG in northern Benin. Tropical Medicine and Health, 52, 18.
Publication 2024
2
CDC Bottle Assay for Clothianidin Susceptibility
The susceptibility of adult mosquitoes was tested against clothianidin using CDC bottle assays [53 ]. The bioassay procedure followed a modified version of the WHO standard operating procedure for testing the susceptibility of adult mosquitoes to clothianidin [54 , 55 (link)]. Precisely, we did not used the vegetable oil ester, Mero®, as a surfactant. Recent studies showed that some vegetable oil-based surfactants such as Mero can enhance the toxicity of neonicotinoids leading to an overestimation of the insecticidal activity of the active ingredient [34 (link), 43 (link), 56 (link)]. Instead, mortality was evaluated against clothianidin alone dissolved in ethanol using a discriminating dose (i.e., the lowest dose at which 100% of adults from a susceptible population die) of 150 µg/ml as determined by a previous study [57 (link)]. We prepared stock solutions using a technical-grade formulation of clothianidin (PESTANAL®, analytical standard, Sigma-Aldrich, Dorset, United Kingdom) and absolute ethanol as solvent. The solutions were stored at 4°C for at least 24 h before use to maximize the solubility of clothianidin.
To perform bottle bioassays, each Wheaton 250-ml bottle and its cap were coated with 1 ml of a solution containing 150 µg/ml clothianidin dissolved in ethanol following the CDC guidelines [53 ]. For each bioassay test, we used four bottles coated with clothianidin and two control bottles treated with 1 ml of absolute ethanol. All bottles were wrapped in aluminum foil and allowed to dry for 24 h to enable complete evaporation of the solvent before use. Coated bottles were not reused and were washed three times in warm soapy water and allowed to dry for 24 h between experiments. 20 to 25 2–5-day-old females were aspired from mosquito cages and released into one of the test bottles where they were exposed to the active ingredient or into control bottles containing ethanol for 1 h. After the exposure period, mosquitoes were transferred into a paper cup and provided with 10% sugar solution. 60-min knockdown rates measured as the mosquito’s inability to move or fly when touched with forceps were scored, and mortality was monitored every 24 h for seven consecutive days. We used adults from two susceptible strains as controls: An. gambiae Kisumu and An. coluzzii Ngousso. Both strains are known to be susceptible to pyrethroid, carbamate, organochlorine and organophosphate insecticides.
Fouet C., Ashu F.A., Ambadiang M.M., Tchapga W., Wondji C.S, & Kamdem C. (2024). Clothianidin-resistant Anopheles gambiae adult mosquitoes from Yaoundé, Cameroon, display reduced susceptibility to SumiShield® 50WG, a neonicotinoid formulation for indoor residual spraying. BMC Infectious Diseases, 24, 133.
Publication 2024
3
Contact Irritancy of IRS Formulations
The WHO cone bioassay is a standard methodology used to assess the residual efficacy of IRS treatments [19 ]. A modified version of this test method using video recordings was performed to compare the contact irritancy of a clothianidin solo formulation and clothianidin-deltamethrin mixture formulations for IRS applied to cement blocks under laboratory conditions. The contact irritancy of a clothianidin solo formulation (SumiShield® 50WG, Sumitomo Chemical Co., Ltd.”) was compared to wettable powder (WP) formulations of clothianidin-deltamethrin mixtures. To control for confounding effects of the pyrethroid, comparison was made to a deltamethrin-only water-dispersible granule IRS formulation. Two types of WP clothianidin-deltamethrin mixtures were tested, one commercially available formulation and one non-commercial generic formulation developed for this study. All commercial products were applied at the label application rate. Untreated cement blocks were used as a negative control. The following five treatments were thus compared in contact irritancy bioassays:

Untreated blocks (control)

Deltamethrin solo formulation WG IRS applied at 25 mg ai/m2

Generic clothianidin-deltamethrin WP IRS applied at 200 mg ai/m2 clothianidin and 25 mg ai/m2 (225 mg ai/m2)

Commercial clothianidin-deltamethrin WP IRS applied at 200 mg ai/m2 clothianidin and 25 mg ai/m2 (225 mg ai/m2)

Clothianidin solo formulation WG IRS applied at 300 mg ai/m2

Syme T., N’dombidjé B., Odjo A., Gbegbo M., Todjinou D, & Ngufor C. (2024). Laboratory evaluation of the contact irritancy of a clothianidin solo formulation vs. clothianidin-deltamethrin mixture formulations for indoor residual spraying against pyrethroid-resistant Anopheles gambiae sensu lato. Parasites & Vectors, 17, 183.
Publication 2024
4
Pyrethroid Resistance Gene Expression in Anopheles gambiae
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The expression level of 13 resistant candidate genes previously reported to be associated with pyrethroid resistance in An. gambiae sl. (CYP6M2, CYP6K1, CYP6Z1, CYP6Z2, CYP4G16, CYP6P1, CYP6P2, CYP6P3, GSTe2, GSTD3, Sap1, Sap2, Sap3) were assessed by a quantitative reverse transcription PCR (qRT-PCR) for one urban site (Fifadji) and one agricultural site (Sèdjè) where the highest resistance level was recorded. As described by Tepa et al. (49) (link), three biological replicates of surviving mosquitoes after exposure to clothianidin (survivors from diluted diagnostic dose) and those unexposed were compared to the susceptible strain An. gambiae Kisumu with two housekeeping genes: Elongation factor (AGAP000883) and Ribosomal Protein S7 (AGAP010592). Total RNA was extracted from 3 batches of 10 mosquitoes each and similarly from the susceptible laboratory strain Kisumu, using the Arcturus PicoPure RNA isolation kit (Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. cDNA (complementary Deoxyribonucleic acid) was synthesized from the purified RNA by reverse transcriptase-PCR using the SuperScript III (Invitrogen, Waltham, MA, USA) and the oligo-dT207 of 19 and RNAse H (New England Biolabs, Ipswich, MA, USA) kit in a total reaction volume of 20 μL. Amplification was performed in an Agilent Mx3005 qRT-PCR thermocycler (Santa Clara, CA, USA) with the following conditions: 95°C for 3 min followed by 40 cycles of 10 s at 95°C and 10 s at 60°C; 1 min at 95°C; 30 s at 55°C and 30 s at 95°C. Samples were amplified in three technical replicates, using three biological replicates for gene expression analysis for each population. MxPRO software (Santa Clara, CA, USA) was used for the calculation of the Ct values for each reaction. Standard curves of assessed genes were established. As the efficiency was different from 100%, the Ct value was adjusted according to efficiency as previously done by Riveron et al. (50) (link). The relative expression was calculated individually according to the 2-DDCT method (51) and plotted with R studio software. Genes were considered as relatively overexpressed to An. gambiae Kisumu when their relative fold changes (FCs) were more than a 2 fold-change (49) (link).
Tchigossou G.M., Dossou C., Tepa-Yotto G.T., Koto M., Atoyebi S.M., Tossou E., Adanzounon D., Gouété M., Sina H., Tchouakui M., Dione M., Wondji C.S., & Djouaka R. (2024). Resistance to neonicotinoids is associated with metabolic detoxification mechanisms in Anopheles coluzzii from agricultural and urban sites in southern Benin. Frontiers in Tropical Diseases, 5.
Publication 2024
5
Insecticide Susceptibility Bioassays of An. gambiae
Susceptibility bioassays were performed according to WHO guidelines [18 ] to assess the susceptibility of the An. gambiae s.l. Covè strain to the AIs in the IRS treatments. Mosquitoes were exposed in tube tests to filter papers impregnated with the discriminating concentration of deltamethrin (0.05%) and in bottle bioassays to the discriminating concentration of clothianidin (4 µg) to assess susceptibility to these insecticides. Further exposures were performed with 5 × and 10 × the discriminating concentration of deltamethrin to assess pyrethroid resistance intensity. To assess synergism and the role of P450s in pyrethroid resistance, mosquitoes were also exposed to the discriminating concentration of deltamethrin (0.05%) with pre-exposure to the cytochrome P450 monooxygenase (P450) inhibitor piperonyl butoxide (PBO) (4%). Insecticide-treated filter papers used for tube tests were obtained from Universiti Sains Malaysia. To prepare test bottles for clothianidin susceptibility bioassays, predetermined quantities of technical grade clothianidin were dissolved in acetone and Mero® (800 ppm concentration) to obtain a 4 µg/ml stock solution. Test bottles were coated by introducing 1 ml of a pre-prepared stock solution into bottles and rotating them using a tube roller before leaving them to dry for 2 h. A total of 100 mosquitoes aged 3–5 days were exposed to each insecticide and dose for 60 min in four replicates of approximately 25. Parallel exposures were performed with PBO alone, silicone oil + acetone-impregnated papers, and acetone + Mero®-coated bottles as controls. Similar numbers of the susceptible An. gambiae s.s. Kisumu strain were exposed to the discriminating concentrations of deltamethrin (0.05%) and clothianidin (4 µg) and appropriate controls to validate the test. Knockdown was recorded at the end of exposure, after which mosquitoes were transferred to untreated containers and provided access to 10% (w/v) glucose solution. Mortality was recorded after 24 h for all exposures. Tests and subsequent mortality recordings were performed at 27 ± 2 °C and 75 ± 10% relative humidity.
Syme T., N’dombidjé B., Odjo A., Gbegbo M., Todjinou D, & Ngufor C. (2024). Laboratory evaluation of the contact irritancy of a clothianidin solo formulation vs. clothianidin-deltamethrin mixture formulations for indoor residual spraying against pyrethroid-resistant Anopheles gambiae sensu lato. Parasites & Vectors, 17, 183.
Publication 2024

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Clothianidin by Merck Group
Sourced in Canada, United States, Germany, France
Clothianidin is a neonicotinoid insecticide used in various agricultural applications. It is a broad-spectrum insecticide that acts as an agonist of the nicotinic acetylcholine receptor in the central nervous system of insects. Clothianidin is commonly used as a seed treatment or soil application to protect crops from a variety of insect pests.
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Imidacloprid by Merck Group
Sourced in Germany, United States, United Kingdom, Sweden, China, Sao Tome and Principe, Italy
Imidacloprid is a chemical compound used in laboratory equipment. It functions as an insecticide, targeting the nervous system of insects. The core purpose of Imidacloprid is to provide a means of pest control in controlled laboratory environments.
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Thiamethoxam by Merck Group
Sourced in United States, Germany, China, United Kingdom, India
Thiamethoxam is a neonicotinoid insecticide developed and manufactured by Syngenta, a division of Merck Group. It is used as a broad-spectrum insecticide in agriculture and horticulture. Thiamethoxam acts as a nicotinic acetylcholine receptor agonist, affecting the central nervous system of insects.
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Clothianidin d3 by Merck Group
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Clothianidin-d3 is a stable isotope-labeled compound used as an analytical standard in the detection and quantification of clothianidin, a neonicotinoid insecticide, in various matrices. It serves as a reference material for analytical methods development and validation.
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Acetamiprid by Merck Group
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Acetamiprid is a laboratory chemical used as an analytical standard for qualitative and quantitative analysis. It is a white crystalline solid that is soluble in organic solvents. Acetamiprid is used as a reference material in the identification and quantification of this compound in various samples.
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Thiacloprid by Merck Group
Sourced in Germany, United States, United Kingdom
Thiacloprid is a neonicotinoid insecticide developed by Bayer CropScience. It is used in agriculture to control a variety of insect pests. Thiacloprid acts as a nicotinic acetylcholine receptor agonist, disrupting the nervous system of targeted insects.
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Imidacloprid d4 by Merck Group
Sourced in United States, Germany
Imidacloprid-d4 is a deuterated analogue of the insecticide Imidacloprid. It is used as an internal standard in analytical methods for the detection and quantification of Imidacloprid in various matrices.
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Thiamethoxam by LGC
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Formic acid by Merck Group
Sourced in Germany, United States, Italy, United Kingdom, France, Spain, China, Poland, India, Switzerland, Sao Tome and Principe, Belgium, Australia, Canada, Ireland, Macao, Hungary, Czechia, Netherlands, Portugal, Brazil, Singapore, Austria, Mexico, Chile, Sweden, Bulgaria, Denmark, Malaysia, Norway, New Zealand, Japan, Romania, Finland, Indonesia
Formic acid is a colorless, pungent-smelling liquid chemical compound. It is the simplest carboxylic acid, with the chemical formula HCOOH. Formic acid is widely used in various industrial and laboratory applications.
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Clothianidin by Fujifilm
Sourced in Japan
Clothianidin is a chemical compound used in various laboratory equipment and procedures. It functions as an insecticide, specifically targeting certain types of insects. The core purpose of clothianidin is to provide a means of controlling and managing insect populations in controlled research environments.

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