AANAT1 regulates insect midgut detoxification through the ROS/CncC pathway

Abstract

Insecticide resistance has been a problem in both the agricultural pests and vectors. Revealing the detoxification mechanisms may help to better manage insect pests. Here, we showed that arylalkylamine N-acetyltransferase 1 (AANAT1) regulates intestinal detoxification process through modulation of reactive oxygen species (ROS)-activated transcription factors cap"n"collar isoform-C (CncC): muscle aponeurosis fibromatosis (Maf) pathway in both the oriental fruit fly, Bactrocera dorsalis, and the arbovirus vector, Aedes aegypti. Knockout/knockdown of AANAT1 led to accumulation of biogenic amines, which induced a decreased in the gut ROS level. The reduced midgut ROS levels resulted in decreased expression of CncC and Maf, leading to lower expression level of detoxification genes. AANAT1 knockout/knockdown insects were more susceptible to insecticide treatments. Our study reveals that normal functionality of AANAT1 is important for the regulation of gut detoxification pathways, providing insights into the mechanism underlying the gut defense against xenobiotics in metazoans.

Fig. 1. AANAT1 gene knockout in B. dorsalis increased susceptibility to insecticide.

a A BdorAANAT1 knockout strain, with 10-bp deletions, was obtained by CRISPR/Cas9. b Confirming BdorAANAT1 knockout efficiency at the protein level by western blot analysis. The samples were extracted from adult midguts. c Susceptibility of the WT and AANAT1^10/10 strain flies’ responses to diverse insecticides by feeding. LC₅₀, lethal concentration that kills 50% of B. dorsalis adults; 95% FL, 95% fiducial limits of LC₅₀. Increased folds in susceptibility were calculated as LC₅₀ of WT/LC₅₀ of AANAT1^10/10. LC₅₀ values were considered significantly different if their 95% FL did not overlap, and asterisks indicate statistical significance of LC₅₀ between the two strains.

Fig. 2. BdorAANAT1 modulates midgut detoxification gene expression.

a Relative expression level of BdorAANAT1 in different gut region of B. dorsalis. b Immunostaining of B. dorsalis gut using BdorAANAT1 antibody. FG, foregut, MG, midgut, HG, hindgut. The scale bar represents 1000 μm. c Principal component analysis of midgut RNA-seq from WT and AANAT1^10/10 strains. R package gmodels was used in this experiment. d Heat map showing the relative expression levels of genes involved in detoxification in the midgut of B. dorsalis. The map is plotted based on Log2-transformed FPKM values, each bar or column corresponds to the relative gene expression level of gene in one sample, with warmer colors representing higher relative gene expression levels. e–t qPCR validation of differentially expressed genes in the midgut of WT and AANAT1^10/10 strains. u GST activity in the midgut of B. dorsalis. v P450 activities were determined using p-nitroanisole-O-demethylase (PNOD) as the substrate. Student’s t test was performed for e–v. Error bars indicate ± s.e.m., ***p < 0.001, **p < 0.01, *p < 0.05.

Fig. 3. Decreasing ROS level by vitamin C treatment affected midgut detoxification in B. dorsalis.

ROS activity of the midgut from WT and AANAT1^10/10 strain flies was measured by H₂O₂ assay (a) and dihydroethidium (DHE) (b). Scale bars in b represent 1000 µm. Data of H₂O₂ level are normalized to WT. c H₂O₂ concentration was determined in the midgut of B. dorsalis after vitamin C treatment. Data of H₂O₂ level are normalized to controls. d, e GST and P450 activities were determined in the midgut of B. dorsalis after vitamin C treatment. f–k Transcriptional responses of LOC109579784 (GstE4), LOC105229682 (GstE7), LOC105229681 (GstE9), LOC105233810 (Cyp6g2), LOC105224660 (UGT36-D1), and LOC115065792 (UGT49-C1) to vitamin C treatment. l Effect of vitamin C treatment on flies to trichlorphon. SS, sensitive strain, VC, vitamin C. Ten-day old adults mixed with the same number of males and females were treated with 100 mg/mL vitamin C for 24 h. Then, flies were treated with trichlorphon or their midguts were dissected on ice. Student’s t test was performed for a, c, d–k. Error bars indicate ±s.e.m., ***p < 0.001, **p < 0.01, *p < 0.05.

Fig. 4. ROS activated CncC: Maf pathway is involved in BdorAANAT1 regulated midgut detoxification process.

mRNA level of Bdor-CncC, Bdor-Maf, and Bdor-Keap1 in the midguts of BdorAANAT1 knockout (a), and knockdown (b) flies. c The effect of vitamin C treatment on the midgut expression of Bdor-CncC, Bdor-Maf, and Bdor-Keap1. d Effect of overexpression of transcription factor BdCncC on the promoter activity of P450 and GST genes. Relative fluorescence (RLU) activity = Firefly luciferase activity/Renilla luciferase activity. e Midgut Bdor-CncC silencing efficiency at 72 h post RNAi. f, g Regulation of both GST and P450 activities in the midguts of CncC-silencing flies. h–v Transcriptional responses of detoxification genes to Bdor-CncC knockdown. Student’s t test was performed. Error bars indicate ±s.e.m., ***p < 0.001, **p < 0.01, *p < 0.05.

Fig. 5. BdorAANAT1 controls ROS production via monoamines in the midguts of B. dorsalis.

Midgut monoamines including serotonin (a), dopamine (b), tyramine (c), and melatonin (d) was determined using HPLC-MS. e Amine mix including serotonin, dopamine, and tyramine inhibited H₂O₂ level in a dose- dependent manner. f The effect of 10 μM serotonin, dopamine, and tyramine on the H₂O₂ level of WT strain. g The effect of 10 μM melatonin on the H₂O₂ level of AANAT1^10/10 strain. Student’s t test was performed for a–f. Error bars indicate ±s.e.m., ***p < 0.001, **p < 0.01, *p < 0.05.

Fig. 6. AANAT1 is involved in midgut detoxification via ROS/CncC pathway in A. aegypti.

a Midgut AANAT1 silencing efficiency determination in A. aegypti by real-time PCR. b Effects of Aa-AANAT1 silencing on midgut H₂O₂ concentration. Data of H₂O₂ level were normalized to controls. c Transcripts level of Aa-CncC, Aa-Maf, and Aa-Keap1 in the midguts of Aa-AANAT1 knockdown mosquitos. d, e GST and P450 activities were determined in the midgut of A. aegypti after dsRNA treatment. f Effect of Aa-AANAT1 silencing on the susceptibility of mosquitos to beta-cypermethrin. LC₅₀ lethal concentration that kills 50% of B. dorsalis adults; 95% FL, 95% fiducial limits of LC₅₀. g Schematic representation of AANAT1 modulates midgut detoxification process. AANAT1 regulates the concentration of gut biogenic amines, which reduce the ROS level. The decreased ROS level inhibited detoxification gene transcription via Cncc:Maf pathway. Decreased detoxification enzyme activity reduced insect survival rate when they are treated with insecticide. Student’s t test was performed for a–e. Error bars indicate ±s.e.m., ***p < 0.001, **p < 0.01, *p < 0.05. LC₅₀ values were considered significantly different if their fiducial limits did not overlap.