Low doses of the organic insecticide spinosad trigger lysosomal defects, elevated ROS, lipid dysregulation, and neurodegeneration in flies
- School of BioSciences, The University of Melbourne, Australia
- Department of Molecular and Human Genetics, Baylor College of Medicine, United States
- Department of Integrative Biology and Pharmacology, McGovern Medical School at the University of Texas Health Sciences Center, United States
- Neurological Research Institute, Texas Children Hospital, United States
- Howard Hughes Medical Institute, Baylor College of Medicine, United States
Figures
Figure 1
Low doses of spinosad are lethal and fail to increase Ca2+ levels in neurons.
(A) Dose–response to spinosad in Line14 wild-type larvae by an assay of larval movement over time, expressed in terms of relative movement ratio (n = 100 larvae/treatment). (B) Adult eclosion rate …
Figure 2
Spinosad exposure causes lysosomal expansion, and Dα6 colocalizes with enlarged lysosomes.
(A) Dα6 signal in larval brains exposed to 2.5 parts per million (ppm) spinosad for 30 min, 1 hr, or 2 hr. Larvae obtained by crossing UAS-Dα6 (CFP tagged) in the Line14 Dα6nx loss-of-function …
Figure 3 with 1 supplement
Spinosad exposure impacts mitochondria and energy metabolism, and antioxidant treatment reduces spinosad toxicity.
(A) Optic lobes of the brain and proventriculus of MitoTimer reporter expressing larvae. 2.5 parts per million (ppm) spinosad exposure for 2 hr increased the signal of healthy (green) and unhealthy …
Figure 3—figure supplement 1
Spinosad exposure increases mitochondrial reactive oxygen species (ROS) signal.
(A) Mito-roGFP2-Orp1 signal in brain and anterior midgut of larvae exposed to 2.5 parts per million (ppm) spinosad for 2 hr. An increase in the signal of both reduced (488 nm – green) and oxidized …
Figure 4 with 1 supplement
Spinosad exposure increases oxidative stress, and antioxidants prevent reactive oxygen species (ROS) accumulation, but not lysosome expansion.
(A) Dihydroethidium (DHE) staining of ROS levels in the brain and anterior midgut of Line14 larvae exposed to 2.5 parts per million (ppm) spinosad for either 1 hr or 2 hr. (B) DHE normalized …
Figure 4—figure supplement 1
Dα6 KO mutants show no increase in ROS levels after spinosad exposure.
(A) Dihydroethidium (DHE) staining of ROS levels in the brains of Canton-S and Canton-S Dα6 KO larvae exposed to 2.5 parts per million (ppm) spinosad for 2 hr. (B) DHE normalized fluorescence …
Figure 5 with 2 supplements
Spinosad triggers reactive oxygen species (ROS)-driven lipid changes in metabolic tissues of wild-type larvae but not Dα6 loss-of-function larvae.
(A) Nile Red staining showing lipid droplets in larval fat bodies of Line14 and Canton-S strains and their respective Dα6 loss-of-function mutant strains. Larvae exposed to 2.5 parts per million …
Figure 5—figure supplement 1
Impact of spinosad exposure on lipid droplets dynamics in fat body.
(A) Number of small (>1.5 µm < 10 µm) and large (10–20 µm) lipid droplets in the fat body of Line14 and Canton-S larvae and respective Dα6 loss-of-function mutants exposed to 2.5 parts per million …
Figure 5—figure supplement 2
Spinosad doses that do not affect survival impact the larval lipid environment.
(A) Corrected adult emergence relative to controls – larvae exposed to different spinosad doses were rinsed in 5% sucrose and placed back onto insecticide-free media for quantification of adult …
Figure 6
Spinosad disturbs the lipid profile of exposed larvae.
Lipidomic profile of larvae exposed to 2.5 parts per million (ppm) spinosad for 2 hr (n = 10 larvae/replicate; three replicates/treatment). (A) 88 lipid species out of the 378 identified were …
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Figure 6—source data 1
Impact of spinosad on the lipidomic profile.
Lipidomic profile of larvae exposed to 2.5 parts per million (ppm) spinosad or control (equivalent dose of dimethyl sulfoxide) for 2 hr as detected by liquid chromatography-mass spectrometry. Values are expressed as peak intensity area normalized to sample weight.
- https://cdn.elifesciences.org/articles/73812/elife-73812-fig6-data1-v1.docx
Figure 7
Chronic effects of spinosad exposure are more severe than loss of Dα6 expression in adult virgin females.
(A) A chronic exposure to 0.2 parts per million (ppm) spinosad kills 50% of flies within 25 days (n = 25 flies/replicate; four replicates/treatment). (B) Chronic exposure to 0.2 ppm spinosad …
Figure 8
Chronic exposure to spinosad causes lipid deposits in retinas.
(A) Nile Red staining of lipid droplets in the retinas of virgin females exposed to 5 parts per million (ppm) spinosad for 6 hr. Cluster of rhabdomeres delimited with yellow dotted line, purple …
Figure 9
Chronic exposure to spinosad impairs the visual system.
(A) Expression pattern of Dα6 in the Drosophila adult female brain (Dα6 T2A Gal4>UASGFP.nls). Detail of the expression in lamina and medulla (optic lobe). OL,-optic lobe; CB, central brain. (B) …
Figure 10
Chronic exposure to spinosad leads to neurodegeneration.
(A–D) Transmission electron microscopy (TEM) of the lamina of virgin females exposed to 0.2 parts per million (ppm) spinosad for 20 days. (A) A regular cartridge of a control fly; blue arrowheads …
Figure 11
Proposed mechanism for internalization of spinosad after binding to Dα6 targets.
(A) Spinosad binds to Dα6 subunit of nicotinic acetylcholine receptors (nAChRs) in the neuronal cell membranes. (B) The binding of spinosad leads to Dα6-containing nAChR blockage, endocytosis, and …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent(Drosophila melanogaster) | Armenia60 | Drosophila Genomics Resource Center | DGRC #103394 | Line14 is an isofemale line derived from Armenia60 |
| Genetic reagent(D. melanogaster) | nAChRα6 T2A Gal4 | Bloomington Drosophila StockCenter | BDSC #76137 | RRID:BDSC_76137 |
| Genetic reagent(D. melanogaster) | UAS-GFP.nls | Bloomington Drosophila StockCenter | BDSC #4775 | RRID:BDSC_4775 |
| Genetic reagent(D. melanogaster) | mito-roGFP2-Orp1 | Bloomington Drosophila StockCenter | BDSC #67672 | RRID:BDSC_67672 |
| Genetic reagent(D. melanogaster) | UAS-Sod2 | Bloomington Drosophila StockCenter | BDSC #24494 | RRID:BDSC_24494 |
| Genetic reagent(D. melanogaster) | UAS-Sod1 | Bloomington Drosophila StockCenter | BDSC #24750 | RRID:BDSC_24750 |
| Genetic reagent(D. melanogaster) | Elav-Gal4 | Bloomington Drosophila StockCenter | BDSC #458 | RRID:BDSC_458 |
| Genetic reagent(D. melanogaster) | Canton-S | Bloomington Drosophila StockCenter | BDSC #64349 | RRID:BDSC_64349 |
| Genetic reagent(D. melanogaster) | Canton-S Dα6 KO;Canton-S nAChRα6 knockout | This paper | Mutant strain generated by CRISPR and maintained in T. Perry Lab | |
| Genetic reagent(D. melanogaster) | Line14 Dα6 loss-of-function mutant;Line14 Dα6nx | Perry et al., 2015 (doi:10.1016/j.ibmb.2015.01.017) | Mutant strain generated by EMS and maintained in T. Perry Lab | |
| Genetic reagent(D. melanogaster) | GCaMP5G:tdTomato cytosolic [Ca2+] sensor | Bloomington Drosophila StockCenter | BDSC #80079 | RRID:BDSC_80079 |
| Chemical compound, drug | Spinosad | Sigma-Aldrich | Product #33706 | |
| Chemical compound, drug | Antioxidant N-acetylcysteine amide;NACA | Liu et al., 2015 (doi:10.1016/j.cell.2014.12.019) | Provided by Hugo J. Bellen Lab | |
| Chemical compound, drug | DHE | Sigma-Aldrich | Product #D7008 | |
| Chemical compound, drug | Nile Red | Sigma-Aldrich | Product #N3013 | |
| Chemical compound, drug | LysoTracker Red DND-99 (1:10,000) | Invitrogen | Cat #L7528 | |
| Commercial assay, kit | Mitochondrial aconitase activity kit | Sigma-Aldrich | Product #MAK051 | |
| Commercial assay, kit | ATP assay kit | Abcam | Product #ab83355 |
