Abstract:  【Aim】 Apolipoprotein D (ApoD) is an extracellular protein involved in various biological functions, including metabolism, tissue development, immunity and antioxidation. It serves as a crucial molecular basis for anti-aging and lifespan extension. This study aims to elucidate the duplication and expansion extent, and phylogenesis of ApoD genes in the genomes of spider mites and explore the impact of multifunctional ApoD genes on the longevity and reproduction of Tetranychus urticae.【Methods】 A combined approach utilizing BLASTP, HMMER, TBLASTN and GEMOMA was employed to identify the members of the ApoD gene family in the genomes of Aculops lycopersici, Tetranychus truncatus and T. urticae. The phylogenetic tree of ApoDs from bacteria, fungi, plants, mammals, insects, gall mites and spider mites was constructed with the maximum likelihood method. Based on the expression profiles of the ApoD family genes of T. urticae in different developmental stages (egg, nymph, 1-day-old female adult and 5-day-old female adult) and nymphs or adults on different host plants (bean, Arabidopsis thaliana, tomato, eggplant, cotton and cucumber), four genes (ApoDR2, ApoD9, ApoD17 and ApoD24) were selected for further RNAi. The RNAi of ApoDR2, ApoD9, ApoD17, and ApoD24 in the newly molted adult females was conducted through immersion in dsRNA, and the survival rate and the daily average number of eggs laid per female within 10 d were monitored. 【Results】 A total of 68 ApoD genes in the T. urticae genome were identified. There were 33 of 68 ApoD genes in the closely related T. truncatus and one in A. lycopersici. Outside the Tetranychidae family, organisms typically possessed 1-10 ApoD genes. Phylogenetic analysis result revealed that the ApoD gene family in spider mites clustered into three major clades, aligning with lipid transport protein genes of insects, gall mites and fungi, respectively. The expanded ApoD lineage of spider mites exhibited multiple unique conserved sites shared with fungal ApoD genes, and the maximum likelihood tree suggested a close evolutionary relationship between them. Most of these ApoD genes exhibited high expression levels in nymph and adult and displayed diverse expression regulation patterns in T. urticae fed on different host plants. The silencing of ApoDR2 and ApoD9 showed no significant impact on the fitness of T. urticae, while the silencing of ApoD17 and ApoD24 significantly reduced the survival rate and daily average number of eggs laid per female of T. urticae, with the silencing of ApoD17 exhibiting greater effects on the survival rate and daily average number of eggs laid per female withing 10 d compared with the control. 【Conclusion】 The ApoD genes, likely acquired from fungal horizontal transfer, underwent substantial expansion in the genomes of spider mites, showing varying degrees of impacts on the longevity and reproduction of T. urticae. However, the multifunctionality of ApoD genes in spider mites requires further investigation.

Key words: Tetranychus urticae, apolipoprotein D, horizontal transfer, gene duplication and expansion, longevity, reproduction