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Int J Mol Sci
2021 Dec 10;2224:. doi: 10.3390/ijms222413291.
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Juvenile African Clawed Frogs (Xenopus laevis) Express Growth, Metamorphosis, Mortality, Gene Expression, and Metabolic Changes When Exposed to Thiamethoxam and Clothianidin.
Jenkins JA
,
Hartop KR
,
Bukhari G
,
Howton DE
,
Smalling KL
,
Mize SV
,
Hladik ML
,
Johnson D
,
Draugelis-Dale RO
,
Brown BL
.
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Neonicotinoids (NEO) represent the main class of insecticides currently in use, with thiamethoxam (THX) and clothianidin (CLO) primarily applied agriculturally. With few comprehensive studies having been performed with non-target amphibians, the aim was to investigate potential biomarker responses along an adverse outcome pathway of NEO exposure, whereby data were collected on multiple biological hierarchies. Juvenile African clawed frogs, Xenopus laevis, were exposed to commercial formulations of THX and CLO at high (100 ppm) and low (20 ppm) concentrations of the active ingredient. Mortality, growth, development, liver metabolic enzyme activity, and gene expression endpoints were quantified. Tadpoles (n > 1000) from NF 47 through tail resorption stage (NF 66) were exposed to NEO or to NEO-free media treatments. Liver cell reductase activity and cytotoxicity were quantified by flow cytometry. Compared to control reference gene expressions, levels of expression for NEO receptor subunits, cell structure, function, and decontamination processes were measured by RT-qPCR by using liver and brain. Mortality in THX high was 21.5% compared to the control (9.1%); the metabolic conversion of THX to CLO may explain these results. The NF 57 control tadpoles were heavier, longer, and more developed than the others. The progression of development from NF 57-66 was reduced by THX low, and weight gain was impaired. Liver reductases were highest in the control (84.1%), with low NEO exhibiting the greatest reductions; the greatest cytotoxicity was seen with THX high. More transcriptional activity was noted in brains than in livers. Results affirm the utility of a study approach that considers multiple complexities in ecotoxicological studies with non-target amphibians, underscoring the need for simultaneously considering NEO concentration-response relationships with both whole-organism and biomarker endpoints.
Figure 1. (A) Average total lengths (SE) of tadpoles (Xenopus laevis) measured incrementally from day 1 to day 44 post-hatch. Tadpoles were exposed in vivaria to thiamethoxam (THX), clothianidin (CLO), or pesticide-free control media. Lengths were measured from photographs of all individuals in each vivarium per treatment group (n = 4 replicate vivaria per treatment group). One asterisk indicates significant difference from control by ANOVA at the day of sampling, and two asterisks indicate significance by ANOVA and MANOVA (days 30 and 44). (B) The difference in tadpole lengths (SE) within treatment groups are plotted relative to control lengths.
Figure 2. The progression of developmental stages of Xenopus laevis tadpoles during metamorphosis from the Nieuwkoop and Faber stages NF 57–66 in thiamethoxam (THX), clothianidin (CLO), or neonicotinoid-free control treatment groups. Each dot is the mean per one of four replicate vivaria. Solid lines were plotted after applying a two-parameter logistic growth model of developmental stage data from NF 57–66. The THX 20 ppm (low concentration) was depressed compared to that of the control group (p = 0.004).
Figure 3. Cumulative mortality at days 44 and 76 of Xenopus laevis tadpoles exposed to thiamethoxam (THX) and clothianidin (CLO) treatments at high (100 ppm) and low (20 ppm) concentrations compared with that from neonicotinoid-free media. Differences among treatment groups at both days were noted, with control treatment mortality lower than THX high (p = 0.0007). Numbers within bars indicate the average mortality percentages. A different letter within a day group implies significance.
Figure 4. Representative flow cytometric cytograms from analysis of liver cells from 5 pooled organs (n = 3 replicates per treatment group) from Xenopus laevis tadpoles following exposure to neonicotinoids. (A) Debris is gated out at the origin, with 31.0% of intact, large cells further analyzed. (B,C) Reductases from cells from thiamethoxam 100 ppm (THX high) and clothianidin at 20 ppm (CLO low) indicate more metabolically active cells apparent in the THX high (63.9%) than the CLO low (37.3%), and higher levels of dead cells in THX high (B,D) with CLO lo showing a predominance of injured rather than dead cells (C). (D,E) Cells in the injured/dead gate are displayed in histogram format with the geometric means of green fluorescence (FL1-H) clearly showing higher values in THX high (1312) than in the CLO low treatment group (144). Data were collected 24 h after tadpoles were removed from the treatment.
Figure 5. Flow cytometric data on the proportion of reductase activity and the cytotoxicity of the dead and injured cell populations from livers of Xenopus laevis tadpoles exposed for 44 days in neonicotinoid-free control media, or in either thiamethoxam (THX) or clothianidin (CLO) treatment groups at 20 and 100 ppm, or low and high, respectively. From 3 to 7 flow cytometric analyses were performed on 5 livers pooled per treatment (n = 24 total analyses). Data were collected 24 h after tadpoles were removed from the treatment and placed into neonicotinoid-free media. Letters represent differences among treatment groups per bar color (reductase activity, p < 0.0001; cytotoxicity, p = 0.0129).
Figure 6. Gene expression levels of target genes muscarinic acetylcholine receptor 4 (chrm4), nicotinic acetylcholine receptor subunit α7 (chrna7), acetylcholinesterase (ache), cytochrome P450 (cyp1a1), and actin (act) in Xenopus laevis tadpoleliver and brain tissues following exposure to thiamethoxam (THX) and clothianidin (CLO) at 20 and 100 ppm, or low and high, respectively, at 44 days. Target gene expressions were normalized to those from tadpoles in the control neonicotinoid-free treatment by using geometric means of both odc and gapdh reference genes; expression of chrm4 was normalized to odc only. Asterisks denote values differing significantly from the pesticide-free treatment per tissue type.
S. Figure S1. Dissecting sedated Xenopus laevis tadpoles at developmental stage NF 57. A) Location of incision point for brain removal is at forceps tips at the center of the head. B) Brain removed is a creamy white mass. C) Liver is removed ventrally, being yellowish brown and attached to the end of the gut.
S. Figure S2. Flow cytometric data on the overall proportion of reductase activity from livers of Xenopus laevis having been exposed for 44 days in neonicotinoid-free control media, and thiamethoxam (THX) and clothianidin (CLO) treatment groups at 20 and 100 ppm, or low and high, respectively. Data were collected at 24 hours (P < 0.0001) and 48 hours (P = 0.0717) after tadpoles were removed from the treatment and placed in media. Data at 24 hours were averaged for analyses performed on four separate dates, with from 3 to 7 analyses per treatment group (n = 24 total analyses) by using 5 livers pooled per treatment. Data analysis at 48 hours were performed on two dates, with 2 analyses per treatment group (n = 10 total analyses).
S Figure S3. Flow cytometric data on the cytotoxicity of the dead and injured cell populations livers of Xenopus laevis having been exposed for 44 days in neonicotinoid-free control media, and thiamethoxam (THX) and clothianidin (CLO) treatment groups at 20 and 100 ppm, or low and high, respectively. Geometric means of the flow cytometrically gated subpopulation was collected at 24 hours (P = 0.0129) and 48 hours (P = 0.4212) after tadpoles were removed from the treatments and placed in media. Data at 24 hours were averaged for analyses performed on four separate dates, with from 3 to 7 analyses per treatment group (n = 24 total analyses) by using 5 livers pooled per treatment. Data analysis at 48 hours were performed on two dates, with 2 analyses per treatment group (n = 10 total analyses).
S Figure S4. The evaluation of potential reference genes by comparing thermal cycle threshold values by rtPCR. Fluorescent signals varied the least for ornithine decarboxylase (odc) and glyceraldehyde-3-phosphate dehydrogenase (gapdh), thus being selected as reference genes. Signals for actin (act) and elongation factor 1-α (efl-a) varied the most. Trend lines are plotted and confidence intervals are shown as dashed lines.
Altschul,
Basic local alignment search tool.
1990, Pubmed
Altschul,
Basic local alignment search tool.
1990,
Pubmed
Anderson,
Neonicotinoids in the Canadian aquatic environment: a literature review on current use products with a focus on fate, exposure, and biological effects.
2015,
Pubmed
Ankley,
Adverse outcome pathways: a conceptual framework to support ecotoxicology research and risk assessment.
2010,
Pubmed
Baker,
A meta-analysis of the effects of pesticides and fertilizers on survival and growth of amphibians.
2013,
Pubmed
Basley,
Neonicotinoids thiamethoxam and clothianidin adversely affect the colonisation of invertebrate populations in aquatic microcosms.
2018,
Pubmed
Benson,
GenBank.
2010,
Pubmed
BOELL,
Cytochrome oxidase activity in mitochondria during amphibian development.
1955,
Pubmed
Boily,
Acetylcholinesterase in honey bees (Apis mellifera) exposed to neonicotinoids, atrazine and glyphosate: laboratory and field experiments.
2013,
Pubmed
Boone,
Effects of carbaryl on green frog (Rana clamitans) tadpoles: timing of exposure versus multiple exposures.
2003,
Pubmed
Bradley,
Expanded Target-Chemical Analysis Reveals Extensive Mixed-Organic-Contaminant Exposure in U.S. Streams.
2017,
Pubmed
Bray,
Mice deficient in CHRNA7, a subunit of the nicotinic acetylcholine receptor, produce sperm with impaired motility.
2005,
Pubmed
Brown,
Acetylcholine and cholinergic receptors.
2019,
Pubmed
Browne,
Reproduction and larval rearing of amphibians.
2007,
Pubmed
,
Xenbase
Caron-Beaudoin,
Effects of Neonicotinoids on Promoter-Specific Expression and Activity of Aromatase (CYP19) in Human Adrenocortical Carcinoma (H295R) and Primary Umbilical Vein Endothelial (HUVEC) Cells.
2016,
Pubmed
Colombo,
Exposure to the organophosphorus pesticide chlorpyrifos inhibits acetylcholinesterase activity and affects muscular integrity in Xenopus laevis larvae.
2005,
Pubmed
,
Xenbase
Cook,
SAS programs for real-time RT-PCR having multiple independent samples.
2004,
Pubmed
Cothran,
Proximity to agriculture is correlated with pesticide tolerance: evidence for the evolution of amphibian resistance to modern pesticides.
2013,
Pubmed
Danis,
Investigating Acute and Subchronic Effects of Neonicotinoids on Northwestern Salamander Larvae.
2021,
Pubmed
de Perre,
Fate and effects of clothianidin in fields using conservation practices.
2015,
Pubmed
Dickinson,
Development of the primary mouth in Xenopus laevis.
2006,
Pubmed
,
Xenbase
Faro,
Clothianidin, a neonicotinoid insecticide, activates α4β2, α7 and muscarinic receptors to induce in vivo dopamine release from rat striatum.
2019,
Pubmed
Feyereisen,
Insect P450 enzymes.
1999,
Pubmed
Finnegan,
Comprehensive characterization of the acute and chronic toxicity of the neonicotinoid insecticide thiamethoxam to a suite of aquatic primary producers, invertebrates, and fish.
2017,
Pubmed
Fulton,
Acetylcholinesterase inhibition in estuarine fish and invertebrates as an indicator of organophosphorus insecticide exposure and effects.
2001,
Pubmed
Gauthier,
State of the art on insect nicotinic acetylcholine receptor function in learning and memory.
2010,
Pubmed
Gavel,
Effects of 2 Neonicotinoid Insecticides on Blood Cell Profiles and Corticosterone Concentrations of Wood Frogs (Lithobates sylvaticus).
2019,
Pubmed
Gavel,
Trematodes coupled with neonicotinoids: effects on blood cell profiles of a model amphibian.
2021,
Pubmed
Gibbons,
A review of the direct and indirect effects of neonicotinoids and fipronil on vertebrate wildlife.
2015,
Pubmed
Glennemeier,
Developmental changes in interrenal responsiveness in anuran amphibians.
2002,
Pubmed
,
Xenbase
Gunderson,
Effect of low dose exposure to the herbicide atrazine and its metabolite on cytochrome P450 aromatase and steroidogenic factor-1 mRNA levels in the brain of premetamorphic bullfrog tadpoles (Rana catesbeiana).
2011,
Pubmed
Gurdon,
Methods for nuclear transplantation in amphibia.
1977,
Pubmed
Han,
Human exposure to neonicotinoid insecticides and the evaluation of their potential toxicity: An overview.
2018,
Pubmed
Hladik,
Widespread occurrence of neonicotinoid insecticides in streams in a high corn and soybean producing region, USA.
2014,
Pubmed
Holtswarth,
Effects of the Neonicotinoid Insecticide Clothianidin on Southern Leopard Frog (Rana sphenocephala) Tadpole Behavior.
2019,
Pubmed
Honda,
Neo-nicotinoid metabolic activation and inactivation established with coupled nicotinic receptor-CYP3A4 and -aldehyde oxidase systems.
2006,
Pubmed
Hrynyk,
Effect of imidacloprid on the survival of Xenopus tadpoles challenged with wild type frog virus 3.
2018,
Pubmed
,
Xenbase
Hua,
Evolved pesticide tolerance in amphibians: Predicting mechanisms based on pesticide novelty and mode of action.
2015,
Pubmed
Iturburu,
Uptake, distribution in different tissues, and genotoxicity of imidacloprid in the freshwater fish Australoheros facetus.
2017,
Pubmed
Iwamoto,
Induction of cytochrome P450 family 1 mRNAs and activities in a cell line from the frog Xenopus laevis.
2012,
Pubmed
,
Xenbase
Karimi,
Xenbase: a genomic, epigenomic and transcriptomic model organism database.
2018,
Pubmed
,
Xenbase
Knapen,
Adverse outcome pathway networks I: Development and applications.
2018,
Pubmed
Lee-Jenkins,
Effects of neonicotinoids on putative escape behavior of juvenile wood frogs (Lithobates sylvaticus) chronically exposed as tadpoles.
2018,
Pubmed
Main,
Widespread use and frequent detection of neonicotinoid insecticides in wetlands of Canada's Prairie Pothole Region.
2014,
Pubmed
Mann,
Amphibians and agricultural chemicals: review of the risks in a complex environment.
2009,
Pubmed
Marshall,
Synaptic localization of alpha-bungarotoxin binding which blocks nicotinic transmission at frog sympathetic neurons.
1981,
Pubmed
Matta,
Nicotinic acetylcholine receptor redux: Discovery of accessories opens therapeutic vistas.
2021,
Pubmed
Mikó,
Age-dependent changes in sensitivity to a pesticide in tadpoles of the common toad (Bufo bufo).
2017,
Pubmed
Miles,
Effects of clothianidin on aquatic communities: Evaluating the impacts of lethal and sublethal exposure to neonicotinoids.
2017,
Pubmed
Morrissey,
Neonicotinoid contamination of global surface waters and associated risk to aquatic invertebrates: a review.
2015,
Pubmed
Padilla,
Zebrafish developmental screening of the ToxCast™ Phase I chemical library.
2012,
Pubmed
Paradis,
APE: Analyses of Phylogenetics and Evolution in R language.
2004,
Pubmed
Pérez-Iglesias,
The genotoxic effects of the imidacloprid-based insecticide formulation Glacoxan Imida on Montevideo tree frog Hypsiboas pulchellus tadpoles (Anura, Hylidae).
2014,
Pubmed
Perrin,
The actin gene family: function follows isoform.
2010,
Pubmed
Pfaffl,
A new mathematical model for relative quantification in real-time RT-PCR.
2001,
Pubmed
Pochini,
Reciprocal effects of pesticides and pathogens on amphibian hosts: The importance of exposure order and timing.
2017,
Pubmed
Qi,
Toxicity assessments with Daphnia magna of Guadipyr, a new neonicotinoid insecticide and studies of its effect on acetylcholinesterase (AChE), glutathione S-transferase (GST), catalase (CAT) and chitobiase activities.
2013,
Pubmed
Regnault,
Unexpected metabolic disorders induced by endocrine disruptors in Xenopus tropicalis provide new lead for understanding amphibian decline.
2018,
Pubmed
,
Xenbase
Regnault,
Impaired liver function in Xenopus tropicalis exposed to benzo[a]pyrene: transcriptomic and metabolic evidence.
2014,
Pubmed
,
Xenbase
Relyea,
An unforeseen chain of events: lethal effects of pesticides on frogs at sublethal concentrations.
2008,
Pubmed
Relyea,
Growth and survival of five amphibian species exposed to combinations of pesticides.
2004,
Pubmed
Rieu,
Real-time quantitative RT-PCR: design, calculations, and statistics.
2009,
Pubmed
Rio,
Purification of RNA using TRIzol (TRI reagent).
2010,
Pubmed
Rios,
Effects of imidacloprid on Rana catesbeiana immune and nervous system.
2017,
Pubmed
Robinson,
Clothianidin alters leukocyte profiles and elevates measures of oxidative stress in tadpoles of the amphibian, Rana pipiens.
2021,
Pubmed
Robinson,
Sublethal effects on wood frogs chronically exposed to environmentally relevant concentrations of two neonicotinoid insecticides.
2017,
Pubmed
Roger,
An evaluation of elongation factor 1 alpha as a phylogenetic marker for eukaryotes.
1999,
Pubmed
Rohr,
A qualitative meta-analysis reveals consistent effects of atrazine on freshwater fish and amphibians.
2010,
Pubmed
Rollins-Smith,
Amphibian immunity-stress, disease, and climate change.
2017,
Pubmed
,
Xenbase
Rollins-Smith,
Metamorphosis and the amphibian immune system.
1998,
Pubmed
Samson-Robert,
Neonicotinoid-contaminated puddles of water represent a risk of intoxication for honey bees.
2014,
Pubmed
Schwen,
Hepatic cytochrome P-450-dependent monooxygenase systems of the trout, frog and snake--I. Components.
1982,
Pubmed
Sievers,
Contaminant mixtures interact to impair predator-avoidance behaviours and survival in a larval amphibian.
2018,
Pubmed
Smalling,
Pesticide concentrations in frog tissue and wetland habitats in a landscape dominated by agriculture.
2015,
Pubmed
Sparks,
Insecticide discovery: an evaluation and analysis.
2013,
Pubmed
Stehle,
Agricultural insecticides threaten surface waters at the global scale.
2015,
Pubmed
Stehle,
Compilation and analysis of global surface water concentrations for individual insecticide compounds.
2018,
Pubmed
Swenson,
Neonicotinoid formaldehyde generators: possible mechanism of mouse-specific hepatotoxicity/hepatocarcinogenicity of thiamethoxam.
2013,
Pubmed
Tennekes,
The molecular basis of simple relationships between exposure concentration and toxic effects with time.
2013,
Pubmed
Thornton,
Real-time PCR (qPCR) primer design using free online software.
2011,
Pubmed
Tomizawa,
Imidacloprid, thiacloprid, and their imine derivatives up-regulate the alpha 4 beta 2 nicotinic acetylcholine receptor in M10 cells.
2000,
Pubmed
Tomizawa,
Neonicotinoid insecticide toxicology: mechanisms of selective action.
2005,
Pubmed
Untergasser,
Primer3--new capabilities and interfaces.
2012,
Pubmed
van der Sluijs,
Conclusions of the Worldwide Integrated Assessment on the risks of neonicotinoids and fipronil to biodiversity and ecosystem functioning.
2015,
Pubmed
Vandesompele,
Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.
2002,
Pubmed
Wang,
Study on neurotoxicity of dinotefuran, thiamethoxam and imidacloprid against Chinese lizards (Eremias argus).
2019,
Pubmed
Whyte,
Ethoxyresorufin-O-deethylase (EROD) activity in fish as a biomarker of chemical exposure.
2000,
Pubmed
