Sexual Transcription Differences,Molecular Clone And Expression Analysis Of Sex Determination Genes In Trichogramma Dendrolimi

Sex determination is one of the necessary processes in development and growth of bisexual organisms.Insect sex determination mechanisms are diverse and complex.The study of sex determination mechanism of important insects is not only of theoretical significance for developmental and evolutionary biology,but also of practical value for the utilization of insect-resources.Trichogramma dendrolimi is an important natural enemy of egg parasitoids,which is widely used to control agricultural and forestry Lepidopteran pests.In field control,only females are capable of parasitism.Therefore,increasing the population’s female ratio will greatly improve the effectiveness of control and reduce production costs.To clarify the sex determination mechanism of T.dendrolimi will be able to manipulate Trichogramma reproduction and change the sex ratio.So far,there are few studies on sex determination of Trichogramma.Under haplodiploidy for the sex of Trichogramma,females are diploid and develop from fertilized eggs,whereas males are haploid and develop from unfertilized eggs.The study used the genome and transcriptome of T.dendrolimi,annotated and cloned the sex determination related genes,and analyzed three key genes(tra,tra2,and dsx)alternative splicing and expression level at developmental stages.The results of study will laid an important theoretical foundation for sex determination mechanism in T.dendrolimi.The results as follow:1.High-throughput sequencing technology was used to sequence and assembly the transcriptomes of female and male of T.dendrolimi,and a total of 120825 unigenes were obtained,with an average length of 2399 bp and an N50 of 4459 bp.By comparative transcriptome analysis,a total of 6130 differentially expressed genes were identified in T.dendrolimi,among which 5248 female biased and 882 male biased genes annotated.The majority of the sex-biased genes were female biased.In GO enrichment analysis,2655 biased genes were enriched in biological processes,875 biased genes were enriched in cellular components,and 4295 biased genes were enriched in molecular functions.The enrichment analysis of KEGG showed that female biased genes mainly enriched in the pathways related to DNA replication,RNA transcription translation,and oocyte formation,whereas male biased genes significantly enriched in the pathways related to growth and development of germ cells.These transcriptome data provide some valuable information for exploring the molecular mechanisms in sex determination and sex difference in T.dendrolimi.2.In the study,the combined genome and transcriptome-wide analyses identified 24 genes putatively associated with sex determination in T.dendrolimi,among them,15 genes involved in somatic sex determination,5 genes involved in dosage compensation sex determination,and 4 genes involved in germ cell sex determination.3.The ORF of Tddsx gene is 768 bp encoding for 735 amino acids.The Tddsx is sex-specifically spliced into one female and one male-specific splice form.The DSX proteins in T.dendrolimi share common DM and Dimer domains and differ in their C-terminal sequences.The phylogenetic analysis showed that the female-specific DSX in T.dendrolimi was closely related to the female-specific DSX proteins from N.vitripennis.The absolute expression level of Tddsx was sex-specific and stage-specific.In fertilized eggs,The absolute expression of Tddsx peaked at 11 h,48h,96 h and 168 h,with the highest expression at 168 h.In unfertilized eggs,The absolute expression of Tddsx peaked at 4h,23 h,and 144 h,with the highest expression at 144 h.The absolute expression of Tddsx was higher in fertilized eggs than in unfertilized eggs.4.The full-length sequence of Tdtra from T.dendrolimi has 1592 bp,an ORF of1128 bp,a 2543-bp 5’-UTR,and a 184-bp 3’-UTR containing a 26-bp poly-A tail.Tdtra is sex-specifically spliced into one female-specific and one male-specific splice form.Alignments of the derived amino acid composition of Td TRA and N.vitripennis TRA showed 41.12% identity.Multiple sequence alignments of TRA proteins from T.dendrolimi and seven insect species revealed a high degree of sequence conservation in HYM and CAM domains.Phylogenetic analysis showed that TRA proteins were clustered within each insect order,and Td TRA was closely related to that from N.vitripennis.The absolute expression of Tdtra increased gradually with the development time.In fertilized eggs,The absolute expression of Tdtra peaked at 11 h,21h,120 h and 168 h,with the highest expression at 168 h.In unfertilized eggs,The absolute expression of Tdtra peaked at 5h,17 h,and 30 h,with the highest expression at male adults.Tdtra was expressed in the early embryo stage,and the absolute expression level in the first 5 hours showed that Tdtra in unfertilized eggs was higher than that in fertilized eggs.These results suggest that Tdtra is passed from mother to offspring.5.The ORF of Tdtra2 gene is 885 bp encoding for 294 amino acids.A single splice form of tra2 was identified in T.dendrolimi.The phylogenetic analysis showed that the female-specific TRA2 in T.dendrolimi was closely related to the female-specific tra2 proteins from N.vitripennis.Tdtra2 is expressed in early embryo.The absolute expression of Tdtra2 increased gradually with the development time.In fertilized eggs,The absolute expression of Tdtra2 peaked at 30 h,96 h,and 144 h,with the highest expression at female adults.In unfertilized eggs,The absolute expression of Tdtra2 peaked at 3 h,and 23 h,with the highest expression at male adults.In conclusion,these results lay a foundation for exploring the sex determination mechanism of egg parasitoids and provide theoretical reference for manipulating Trichogramma reproduction in the future.