AANAT1 regulates insect midgut detoxification through the ROS/CncC pathway.

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.

Characterization of an agmatine N-acetyltransferase from Bactrocera dorsalis that modulates ovary development.

Agmatine N-acetyltransferase (AgmNAT), which catalyzes the formation of N-acetylagmatine from acetyl-CoA and agmatine, is a member of the GCN5-related N-acetyltransferase family. So far, knowledge of the physiological roles of AgmNAT in insects is limited. Here, we identified one gene encoding protein homologous to that of Drosophila AgmNAT using sequence information from an activity-verified Drosophila AgmNAT in a BLAST search of the Bactrocera dorsalis genome. We expressed and purified B. dorsalis AgmNAT in Escherichia coli and used the purified enzyme to define the substrate specificity for acyl-CoA and amine substrates. Our application of the screening strategy to BdorAgmNAT led to the identification of agmatine as the best amine substrate for this enzyme, with the highest kcat/Km value. We successfully obtained a BdorAgmNAT knockout strain based on a wild-type strain (WT) using the CRISPR/Cas9 technique. The ovary development of the BdorAgmNAT knockout mutants was delayed for 10 days compared with the WT specimens. Moreover, mutants had a much smaller mature ovary size and laid far fewer eggs than WT. Loss of function of BdorAgmNAT caused by RNAi with mature WT females did not affect their fecundity. These findings indicate that BdorAgmNAT is critical for oogenesis. Our data provide the first evidence for AgmNAT in regulating ovary development.

Arylalkylamine N-acetyltransferase 1 gene (AANAT1) regulates cuticle pigmentation and ovary development of the adult oriental fruit fly, Bactrocera dorsalis.

The arylalkylamine N-acetyltransferase (AANAT) enzymes catalyze the acetyl-CoA-dependent acetylation of an amine or arylalkylamine, which is involved in important biological processes of insects. Here, we carried out the molecular and biochemical identification of an arylalkylamine N-acetyltransferase (AANAT) from the oriental fruit fly, Bactrocera dorsalis. Using a bacterial expression system, we expressed and purified the encoded recombinant BdorAANAT1-V3 protein. The purified recombinant protein acts on a wide range of substrates, including dopamine, tyramine, octopamine, serotonin, methoxytryptamine, and tryptamine, and shows similar substrate affinity (i.e., Km values: 0.16-0.26 mM) except for serotonin (Km = 0.74 mM) and dopamine (Km = 0.84 mM). Transcriptional profile analysis of BdorAANAT1 revealed that this gene is most prevalent in adults and abundant in the adult brain, gut, and ovary. Using the CRISPR/Cas9 technique, we successfully obtained a BdorAANAT1 knockout strain based on a wild-type strain (WT). Compared with the WT, the cuticle color of larvae and pupae is normal; however, in adult mutants, the yellow region of their thorax is darkly pigmented, and two black spots were evident at the abdomen's end. Moreover, the female BdorAANAT1 knockout mutant had a smaller ovary than the WT, and laid far fewer eggs. Loss of function of BdorAANAT1 caused by RNAi with mature adult females in which the reproductive system is fully developed had no effect on their fecundity. Altogether, these results indicate that BdorAANAT1 regulates ovary development. Our findings provide evidence for the insect AANAT1 modulating adult cuticle pigmentation and female fecundity.