蛔虫全发育阶段单细胞图谱揭示了调控蛔虫行为的关键的温度感受器Twk-24a

[Objective]The host range of Ascaris,encompasses most vertebrates,including humans.Their eggs and larvae can rapidly initiate development and subsequently infect hosts upon stimulation by appropriate temperatures.Environmental temperature(≥25℃)serves as a crucial factor in inducing larval development.However,the molecular mechanisms underlying how Ascaris sense temperature signals and regulate larval behavior remain unclear.Elucidating these mechanisms could provide novel molecular targets for establishing temperature-based control strategies aimed at preventing early transmission of Ascaris. [Method]This study employs single-cell sequencing technology to construct a developmental atlas of the Ascaris suum,including its dormant phase,eight developmental stages(from one-cell to L2/L3 larvae),the in vivo infection stage(L4 larvae),and adult tissues.Through annotation,various cell populations and differentiated cell types in the embryo were identified.A set of differentially expressed genes was determined by comparing cell types before and after typical temperature fluctuations,specifically from the dormant phase(＜15℃)to the early developmental stage(≥28℃).Electrophysiological experiments were conducted to assess the temperature sensitivity of target genes,leading to the identification of A.suum temperature sensor.Functional experiments using feeding methods and RNA interference(RNAi)on L2 larvae were performed to verify whether silencing temperature sensor affects the response of L2 larvae to different temperatures,as well as their vitality and developmental rate under varying thermal conditions. [Result]In this study,a total of 233,826 cells were filtered and annotated,resulting in the identification of 58 distinct cell types or states.Pseudotime analysis was conducted to map the differentiation trajectories of the early embryonic development in A.suum,revealing that the gastrula and L2 larval stages are crucial periods for organogenesis and infectivity.Differential expression analysis across a temperature between 4℃and 30℃,combined with gene function enrichment and electrophysiological experiments,led to the identification of the temperature-sensitive ion channel gene Twk-24a,which exhibits temperature sensitivity within the range of 25℃to 37℃.RNA interference(RNAi)experiments targeting the L2 larval stage using dsRNA probes designed for the Twk-24a demonstrated that inhibition of Twk-24a effectively reduced larval viability. [Conclusion]This study elucidates the gene transcription profiles of whole developmental stages of A.suum by constructing a comprehensive single-cell developmental atlas.Through screening and identification,the temperature-sensitive ion channel gene Twk-24a in A.suum was isolated and its crucial role in A.suum development under high temperature conditions was revealed.Further experiments demonstrated that inhibiting Twk-24a significantly reduced the larvae's sensitivity to temperature changes,accompanied by decreased survival ability and loss of potential infectivity.It elucidates the specific biological significance of the A.suum's developmental process involving"temperature-sensor-larval behavior"which holds significant value for studying the environmental adaptability of Ascaris.This study also provides a potentially effective molecular target for the prevention and control of ascariasis through temperature manipulation,offering crucial insights for the global management of soil-transmitted helminth infections.