Preliminary Research On The Molecular Mechanism Of Juvenile Hormone Regulation Of Lifespan In Silkworm

Lifespan refers to the period from birth through growth, development, maturation, aging to death. Usually, age is used as a measure of lifespan scale. Lifespan has been studied in many different species(yeast, nematodes, fruit flies, bees, mice and nonhuman primates and even humans, etc.) and at different levels(populational, individual, cellular and moleculer, etc.) broardly and in-depth throughout the world. Although research has made many significant breakthroughs and significant progress, there are still many limitations. In order to reveal the molecular mechanism of regulation of lifespan from a broader perspective, researchers need some new experimental animals. Silkworm is not only the representative of Lepidoptera, but also a model insect, with many strains, clear genetic background, body size is moderate and easily to operated, and a solid research basis and high level platform. Although silkworm used for lifespan research is rarely reported, based on the above advantages, the silkworm could be usd as a potential experimental animal for lifespan research. Lifespan is a relatively complex biological phenomena or trait which is determined or influenced by many factors. After years of research, many considerable progresses have been made in the study of the influencing factors and regulatory mechanisms of lifespan, including the discovery of insulin/insulin-like growth factor 1 signaling and many other classical signaling pathways. Among the factors in the regulation of lifespan, endocrine is particularly important. According to the previous reports, in higher animals, sex hormones(such as testosterone and estrogen), dehydroepiandrosterone(DHEA), growth hormone and insulin could regulate lifespan. In insects, the two most important hormones are juvenile hormone(JH) and 20-hydroxyecdysone(20E). At present, researchers all over the world are focusing on the effects of juvenile hormone in the regulation of the insect growth, development, metamorphosis, reproduction, and other life processes, however, the present study was to investigate the juvenile hormone regulation of silkworm lifespan. The main results and conclusions obtained are as follows: 1. Effects of juvenile hormone content on the lifespan of ilkwormMany previous studies suggested that larvae treatment with juvenile hormone analogues existed more larval instars. But the impact of the JH on adult silkworm has not been reported. In this study, juvenile hormone III(JH III) or JH analogue(methoprene) was injected to the newly eclosion silkworms, we found that the males’ lifespan are significantly reduced, while the lifespan of the females were not significantly affected. By injection of allatostatin to the newly emerged moth to reduce juvenile hormone content, we found that the lifespan of the male moths have been significantly prolonged. Together with this group of positive and negative experiments, we demonstrated that increased juvenile hormone content can shorten the males’ lifespan and decreased juvenile hormone content could significantly prolong the males’ lifespan. 2. Identification of the juvenile hormone binding protein(JHBP) gene family in silkworm and functional analysis of Bm TO geneBy bioinformatic analysis, we analyzed the juvenile hormone binding protein(JHBP) gene family, and identified 30 JHBP genes in silkworm. In the phylogenetic analysis of Bombyx mori, Drosophila melanogaster, Anopheles gambiae and Apis mellifera, we found that all JHBP genes could be clustered into a total of 10 clades from A to J. Among them, most of the genes within the same species may be clustered into a single clade, while other orthologs of different species can be clustered together. Chromosomal localization results showed that most of silkworm genes are distributed as a cluster in chromosome 15 or chromosome 23, while other genes are individually distributed on a particular chromosome. Tissue expression pattern analysis of the fifth larval instar day three showed that the vast majority of genes are highly expressed in head, integument, midgut, Malpighian tubules and gonads. At the same time, BGIBMGA003345 gene in silkworm is highly orthologous to the Drosophila takeout gene. Therefore, we designated the BGIBMGA003345 gene as Bm TO gene. And, we also found that Bm TO is located on chromsome 15, and is highly expressed in head and gonads(testes and ovaries). 3. Impact of juvenile hormone on Bm Fox O expressionFox O gene is a key regulator of lifespan, and a number of regulatory signaling pathways are gathered here. In this study, RT-q PCR was employed to determine the Bm Fox O expression after the males injected with JH III for 24 h, and we found that its expression is significantly reduced. Furthermore, silkworm ovary cell line Bm NS were cultured in the medium with 5 μM, 10 μM and 20 μM of JH III for 6 h, 12 h and 24 h, respectively, we found that the amount of Bm Fox O gene expression was not affected significantly after treatment for 6 h, while the Bm Fox O expression is reduced significantly for 12 h, and then back to normal levels after 24 h. The results showed that the JH III inhibited the expression of Bm Fox O after treated with JH III in Bm NS cells. It also suggested that the response of Bm Fox O expression to JH III in Bm NS cells is in a time and dose dependent manner. 4. The functional analysis of Bm TO geneDrosophila takeout gene regulates feeding behavior, response to circadian rhythms, courtship behavior, and response to starvation and other functions, while the takeout gene is also a convergent point of many regulatory pathways of lifespan. In this study, we used bioinformatics to identify and clone Bm TO gene in silkworm, and found that Bm TO is not only in response to starvation, but also exists the capability of juvenile hormone binding. By overexpressing and RNAi of Bm Fox O in Bm NS cell line, we found that Bm TO expression is upregulated or downregulated, respectively. Furthermore, bioinformatic analysis revealed the presence of Bm Fox O binding sites in the upstream regulatory region of Bm TO gene. Therefore, we speculated that Bm TO gene is one of the downstream targets of Bm Fox O and can be directly transcriptionaly regulated.In summary, in this study, we found that JH can shorten the lifespan of the adult male silkworm, and initially revealed its regulatory mechanism. Combined with previous studies, we hypothesized that JH could combined to its receptor Met, then to its adaptor Tai and other proteins to further form a complex, and subsequently bonded to the JH response element in the upstream regulatory region of Bm Fox O to inhibit its expression, while the lowered Bm Fox O expression induced the reduced Bm TO expression, and thus weaken the ability of the JH binding of the Bm TO, and ultimately shorten the lifespan of the adult males. This study preliminary revealed the molecular mechanism of the lifespan regulation by JH and provided some new insights and ideas for the JH physiological function and molecular mechanism of lifespan regulation.