Evaluation of the Intergenerational Effects of Arecoline on <i>Aedes aegypti</i> via Acute Exposure and Its Role in Inducing the Leg Deformities Involved in Chitin Synthesis Inhibition

Arecoline is a major active alkaloid in Areca catechu L. Currently, its biological toxicity has been extensively explored using multiple models, such as cell lines, animal embryos and representatives of rodent and non-rodent species. However, the intergenerational impacts of this compound on aquatic invertebrate species remain still unclear. Thus, in this study, using Aedes aegypti as the model insect, we evaluated the toxicity of arecoline to the larvae and its subsequent effects on the developmental parameters of the parental (F₀) and offspring (F₁) generations. Arecoline exhibited a high toxicity to A. aegypti larvae with the LC₅₀ value of 72.68 μg/mL via acute exposure for 48 h. As a result, the cumulative pupation rate of F₀ decreased significantly by ranging from 12.96% to 38.50% with the increase of arecoline concentration from 51.63 to 63.27 μg/mL as compared with that of the blank control. Similarly, the F₀ cumulative emergence rate and the average egg number per female exhibited reductions of 15.30%-43.88% and 26.32%-45.10%, respectively. Meanwhile, leg deformities of F₀ adults were observed as the main visible morphological defects with a maximum rate value of 11.89% at 63.27 μg/mL of arecoline. In contrast, the lifecycle parameters for F₀ mentioned above were not significantly affected in the F₁ generation. In addition, Gene Ontology (GO) analysis of the transcriptome data demonstrated that arecoline-induced leg deformities were associated with extracellular chitin-related biological processes. Furthermore, five chitin synthesis pathway genes (G6PI, GFAT, GNPNA, UAP and CHS) of F₀ adults were significantly down-regulated with ranging from 1.62 to 7.80 folds of the control adults. Correspondingly, the activity of chitin synthetase was significantly inhibited by 72.88% after arecoline exposure. As expected, the chitin contents of leg-deformed adults and their legs were also evidently reduced by 61.13% and 65.44%, respectively. These results imply that acute exposure of arecoline is of high toxicity to A. aegypti larvae and affected the subsequent intergenerational growth and development of F₀ rather than F₁, and that a decrease in chitin synthesis was involved in arecoline-induced leg deformities of A. aegypti.