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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.
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