Zebrafish (AB line) were provided by the Inner Mongolia Key Laboratory of Toxicant Monitoring and Toxicology and raised in a circulating aquaculture system (Beijing Aisheng Science and Technology Development Co., Ltd.). The rearing conditions were as follows: the male and female fish were housed in separate tanks and fed worms twice a day in the morning and evening; the temperature was maintained at 28.0–28.6°C under a light-dark cycle of 14:10 h; the pH remained between 7.0 and 7.6 and the conductivity remained between 440 and 640 μs. When breeding, the male and female adults were combined in a 2:1 ratio. The embryos were collected and washed the next morning. Then, embryo selection was performed under a Leica stereomicroscope (Leica, M205, Singapore).

Deltamethrin, (C₂₂H₁₉BrNO₃, purity >99.2%) was purchased from J&K Scientific (Beijing, China). Acetylcholinesterase activity assays were purchased from Solaibo Technology Co., Ltd. (Beijing, China). An ACh Content Test Kit, and BCA Protein Quantitation Kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Then, dimethyl sulfoxide (DMSO), total RNA extraction reagents, and other reagents were purchased from Sigma (St. Louis, MO, United States). The reverse transcription reagents were purchased from Takara (Kusatsu, Japan). A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining kit was purchased from Beyotime Reagent Co., Ltd. (Beijing, China).

Zebrafish embryos (2–4 hpf, hours post fertilization) were selected and exposed with/without DM (0.1% DMSO and 0.05 μg/L, 0.5 μg/L or 5 μg/L) up to 120 hpf, and there were 10 embryos per group and three parallel replicate groups. Every 24 h, the morphological changes were detected, such as the mortality, hatching rate, and deformity rate, and the heart rate was detected at 72, 96, and 120 hpf. Moreover, behavioral tests were conducted at 120 hpf and the embryos were collected for tissue sections and the detection of gene and protein levels.

For the coiling frequency detection, the zebrafish embryos were exposed with/without DM (0.1% DMSO and 0.05 μg/L, 0.5 μg/L, or 5 μg/L) from 2–4 until 32 hpf following the method of Chen et al. (22). The coiling frequency of the zebrafish embryos was recorded for 1 min every 2 h from 18 to 32 hpf under a Leica stereomicroscope. There were 10 zebrafish larvae in each group and three parallel experimental groups.

For the 120 hpf zebrafish behavioral assays, the zebrafish embryos were exposed with/without DM (0.1% DMSO and 0.05 μg/L, 0.5 μg/L, or 5 μg/L) from 2–4 until 120 hpf with the Viewpoint Zebra Lab Behavior System (Viewpoint, Civrieux, France) according to the method of Zhou et al. (23). For each experimental group, 24 well-developed zebrafish larvae without deformity were selected. Subsequently, the zebrafish larvae were placed in a 24-well plate that was filled with 2 mL of fish solution per well. Then, we waited 10 min for zebrafish acclimatization, and the fish were subjected to 10 min of alternating light and dark conditions to detect any zebrafish behavioral changes for 60 min in total. The output consisted of one data point per minute for the statistical analysis.

When the zebrafish embryos developed to 120 hpf, the zebrafish larvae were collected, immobilized with 4% PFA, washed with PBS, dehydrated with a series of alcohols, and paraffin-embedded. Then, 3 μm sections were cut, the sections were dewaxed twice in xylene at 10 min/time, and they were dehydrated with a series of alcohols. After washing with water, the sections were digested with protein kinase K at 37°C for 15 min and PBS was washed twice for 5 min each time. Next, according to the instructions, TUNEL fluorescent dye (Beyotime Reagent Co., Ltd.) was dropped onto the sections, and they were placed in a thermostatic oven at 37°C in the dark. Subsequently, the sections were washed with PBS, dried, and sealed.

The zebrafish embryos (2–4 hpf) were selected and exposed with/without DM (0.1% DMSO and 0.05 μg/L, 0.5 μg/L or 5 μg/L) up to 120 hpf with 10 embryos per group, and three parallel replicate groups. The zebrafish larvae were collected at 120 hpf, and the expressions of myh6, myh7, chrnb3b, and ache messenger RNA (mRNA) were quantified. According to the manufacturer’s instructions, the total RNA was extracted with Trizol reagent. Then, the RNA concentrations were measured using the NanoDrop ND-100 spectrophotometer (Thermo Fischer Scientific, Waltham, MA, United States), which was used to ensure that the 260/280 ratios were greater than 1.8. Next, high-capacity complementary DNA (cDNA) reverse transcription kits (Takara) were used for cDNA generation. Subsequently, 0.25 ng of cDNA was used for a one-step reverse transcription polymerase chain reaction (PCR) reaction (Takara). The samples were analyzed using the Plus One Real-time PCR system (AB Biosynthesis). Then, myh6, myh7, chrnb3b, and ache were studied, and gapdh was used as an internal reference. The primer sequences are presented in Table 1, and the relative gene expression was calculated using the 2 − Δ Δ C T method.

Zebrafish embryos were treated with 0.1% DMSO and 0.5 μg/L or 5 μg/L DM, and transcriptomic analysis was performed in the range of 2–3 to 120 hpf, with 15 zebrafish in each treatment group. There were 3 replicates in each group, and total RNA was extracted with Trizol reagent (n = 3). RNA sequencing (RNA-SEQ) was then performed by Novo Ho (Tianjin, China). Genes with adjusted p-value (padj) <0.05 and fold change >1.5 were defined as differentially expressed genes (DEGs). Next, the Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to analyze the major enrichment pathways of differential genes using R 3.2.2. And the Novo Ho digital analysis tool was used for Gene Ontology (GO) enrichment analysis. Significantly enriched GO terms These GO terms can analyze gene changes in biological processes (BP), cell components (CC) and molecular functions (MF). The corrected p-value (padj) <0.05 was significant.

The zebrafish larvae were treated with/without DM from 2–4 until 120 hpf, there were 120 larvae per group with three parallel replicates for each concentration group. The 120 hpf zebrafish larvae were homogenized to extract the proteins.

According to the method of Chen et al. (24), the detection of the AChE activity and ACh content were carried out. The 120 hpf zebrafish larvae were homogenized to extract the proteins, and detection of the AChE activity and ACh content was performed (Solelbro Reagent). The determination of the AChE activity was based on a chromogenic reaction that converts ACh to the blue product trinitrobenzene. Detection of AChE activity was conducted under a 412 nm light wavelength, and then the AChE activity was calculated. The detection of the ACh content was conducted under 550 nm light wavelength, and then the content of ACh was calculated.

According to the methods of Chen et al. (24), AChE mRNA (BC163898.1) was produced by Bgi Tech Solutions Co., Ltd. (Beijing, China), amplified, and synthesized for gene overexpression. The AChE-MO was synthesized by Gene Tools, LLC (United States) (5′-GGTCTTCATGGCTTCTTTTCACTTG-3′). The best 1–2 cell stage zebrafish embryos were selected for microinjection, and each fish egg was injected with 3 nL. Then, l h after injection, the embryos in good condition were examined and selected for the experiments, and they were placed in an incubator at 28°C for culturing. All the embryos or larvae were observed under the microscope daily, and the dead embryos or larvae were removed.

The experimental data were analyzed and displayed using GraphPad Prism 5.0 (GraphPad, California, United States), and the data were expressed as the mean ± standard error. A one-way analysis of variance and Turkey multiplex test were used to analyze the significance of the differences between groups, and p < 0.05 was considered a significant difference.

Zebrafish embryos were exposed to 0.1% DMSO and 2–4 hpf to 120 hpf of 0.05, 0.5, 5, and 50 μg/L DM, and survival, hatching, and malformation of zebrafish were observed and recorded every 24 hpf. The results showed that from 24 hpf to 120 hpf, 0.05 and 0.5 μg/L DM had no significant effects on the mortality and hatchability of zebrafish (p > 0.05) (Figures 1A,B). However, 50 μg/L DM caused a significant increase in zebrafish mortality, and at 72 hpf DM with an LC50 of 12.32 μg/L DM also caused significant deformities, such as pericardial edema and spinal curvature. Compared with the control group, the deformity rate was increased by 3.50 times (p < 0.05) (Figure 1C). DM also caused significant deformities, mainly manifested as trunk curvature, pericardial edema, and shortened body length, among which pericardial edema was the most significant (Figures 1D–F).

Zebrafish embryos were exposed to 0.1% DMSO and 2–4 hpf to 120 hpf at 0.05, 0.5, 5, and 50 μg/L DM, at which the development of the zebrafish swim bladder and heart sac was measured and recorded (Figure 2). The results showed that with the increase of exposure concentration, the development of swim bladder of zebrafish in DM exposed group was inhibited. When zebrafish developed to 120 hpf, the swim bladder area of zebrafish in 0.5 and 5 μg/L DM groups was reduced by 30.63 and 48.65%, respectively, compared with the control group (Figure 2E). In contrast, the cardiac sac area increased with the increase of exposure concentration. At 120 hpf, the heart sac area of zebrafish in 0.05, 0.5 and 5 μg/L DM groups increased by 1.12, 1.03 and 1.01 times, respectively, compared with the control group (Figure 2F).

To detect the effect of DM on behavioral changes in early zebrafish embryos, 2–4 hpf zebrafish embryos were exposed to 0.1% DMSO and 0.05, 0.5, 5, and 50 μg/L DM, and the frequency of embryo coiling was detected at 18–32 hpf (Figure 3). When the zebrafish embryo developed to 18 hpf, the zebrafish embryo began to show coiling movement, but DM had no significant effect on the curling movement. However, at 20–22 hpf, 0.05, 0.5, and 5 μg/L DM significantly increased the frequency of embryo coiling by more than 1.45 times compared to the control group (p < 0.001). At 24 hpf, the crimp frequency of 0.5 μg/L DM group was significantly increased by 1.51 times compared with the control group (p < 0.05). However, 5 μg/L DM did not significantly increase the crimp frequency. At 26–32 hpf, the coiling frequency decreased gradually, but DM exposure had no significant effect on the coiling frequency (p > 0.05). 0.05 μg/L DM exposure had no effect significantly on the curled frequency of 18–32 hpf zebrafish embryos (p > 0.05).

When detecting the effect of AChE overexpression or AChE knockdown on the behavioral changes in the early zebrafish embryos, the zebrafish embryos (2–4 hpf) were injected with AChE-MO and AChE overexpression and exposed to DM. The frequency of the coiling movement of the embryos was detected at 18–32 hpf (Figure 3). When the zebrafish embryos were at 18 hpf, the coiling movement began to appear, but DM had no significant effect on the coiling movement. However, at 20–22 hpf, 0.5 μg/L DM significantly increased the frequency of the coiling movements, and they reached their highest value relative to that of the control group, with an increase of more than 1.45 times (p < 0.05). At 22 hpf, the coiling frequency of the embryos which were injected with AChE overexpression and exposed to 0.5 μg/L DM significantly increased than that in the 0.5 μg/L DM group (p < 0.01). At 24–32 hpf, the coiling frequency gradually decreased in each group; when contrasted with the control group, the other four groups did not have significantly affected coiling movements (p > 0.05).

When the embryos were at 120 hpf and became larvae, the changes in the swimming speed of the zebrafish larvae were detected under alternating light and dark conditions, and the change index of the one-minute swimming distance was quantitatively analyzed (Figure 4). Specifically, 0.5 μg/L DM and 5 μg/L DM and AChE MO increased the swimming speed of the zebrafish larvae under both light and dark conditions in relation to the control group, and the average swimming speed increased by 1.33–2.12 times (p < 0.05). In both the light and dark environments, the swimming speed of the embryos that were microinjected with AChE and exposed to the 5 μg/L DM group was significantly reduced 56.95 and 45.67% when compared to that of the 5 μg/L DM group (p < 0.05; Figures 4A–D).

The zebrafish larvae underwent one-step TUNEL staining at 120 hpf (Figures 5A–F). The TUNEL-positive cells were observed in the brain of the zebrafish larvae (Figures 5D–F). The 0.05 and 0.5 μg/L DM treatment significantly increased the number of TUNEL-positive cells. When compared with the control group, the number of TUNEL-positive cells in the 0.05 and 0.5 μg/L DM groups increased by 1.65 and 1.82 times respectively, (p < 0.05; p < 0.01; Figure 5G).

The zebrafish embryos were exposed to DM for detection of the AChE gene (ache), ACh gene (chrnb3b), and vascular smooth muscle associated genes (myh6 and myh7) at 4 hpf to 120 hpf. At 120 hpf, the ache, chrnb3b, myh6, and myh7 mRNA expression for 0.05 and 0.5 μg/mL DM was compared (Figure 9A). When compared with the control group, 0.05 and 0.5 μg/L DM ache mRNA expression decreased by 47.08 and 56.15% (p < 0.05; Figure 9A), chrnb3b increased by 1.23 times and 1.82 times (p < 0.05; Figure 9B), myh6 mRNA expression decreased by 28.53 and 65.56% (p < 0.05), and myh7 mRNA expression increased by 1.76 times and 1.56 times (p > 0.05; Figures 9C,D), respectively.

Whether DM exposure affects the changes in AChE activity and the ACh content was determined (Figure 10). When compared with the control group, 0.05 and 0.5 μg/L DM reduced the AChE activity by 27.3 and 59.7% (p < 0.05; Figure 10A), respectively. Correspondingly, the ACh content increased significantly in comparison with the control group, increasing by 1.44 and 2.05 times, respectively (p < 0.05; Figure 10B).

Zebrafish embryos were injected with AChE mRNA (AChE overexpression) or AChE-MO (AChE knockdown) at the 1–2 cell stage. After DM exposure, the larvae in each group were collected at 120 hpf, and changes in their AChE activities and ACh content were detected. When compared with the control group, AChE-MO reduced the AChE activity by 66.1% (p < 0.05), and AChE overexpression increased the AChE activity by 1.76 times (p < 0.05). When AChE overexpression was exposed to 0.5 μg/L DM, it reduced the AChE activity by 69.1% (p < 0.05). Correspondingly, when AChE overexpression was exposed to 0.5 μg/L DM it was reduced by 20.9% (p < 0.05; Figure 10B).