| The seabuchthorn carpenterworm (Holcecerus hippophaecolus Hua. Chou, Fang et Chen)(Lepidoptera:Cossidae) is a native borer pest in China. Its outbreak has been reported in recent years, which is considered to be a main threat to the seabuckthorn (Hippophae rhamnoides L.). Researches on H. hippophaecolus involving the mechanism of outbreak, biological characteristics, as well as control methods were well studied in the past years. This paper examined the basic reproductive endocrinology system of H. hippophaecolus. The molecular biology and hormone detennination method was used to study the regulation characteristics of the Holcocerus ecdysone, Halloween gene, the ecdysone receptor EcR, and the diapause hormone receptors DHR in different stages of development. In addition, the morphological and histological methods was used to compare the differences of the Holcocerus reproductive system between pre-and post-mating. The main conclusions are as follows:1. The extraction and quantitative method of identifying carpenterworm20-hydroxyecdysone titers by liquid chromatography-mass spectrometry was successfully established. The results showed that the body weight and body length of H. hippophaecolus increased significantly from the12th-16th larval instar. In assumption, the first two years’life history aims to keep a stable physiological activity level, while cell differentiations occurs during the third year is to prepare for the tissues and organs restructuring of pupal stages. The small peak of20E in6-day-old last instar larval corresponded to the reorganization, and small peak of pupal stage corresponded to the complete of reorganization, the major peak of20E in2-day-old female adults corresponded to the high activity of mating and spawning. Therefore, the results surported our hypothesis, and20E did play an important role in regulating the Holcocerus adult reproductive process in our study.2. The molecular characteristics and expressions of Holcocerus CYP307A1(spook), CYP306A1(phamtom) and CYP414A1(shade) was identified. These three genes exhibited the conserved P450domain. The expression of HhSpo in last larval instar and adult was consistent in20E level. HhSpo which involved in ecdysone synthesis in the entire growth and reproductive processes had no paralogs. In addition, the spatial and temporal dynamic expression of HhSpo, HhPhm and HhShd indicated that those three genes had different effect on maintaining the20E level in hemolymph. Finally, these three genes and20E co-regulate each other during the development of Holcocerus.3. The characteristics and expression of Holcocerus EcR were identified. HhEcR was mainly expressed in fatbody and prothoracic gland of the last larva and ovary of femal adult. In the early stage of the last instar larvae, lower20E titer increased the transcript level of HhEcR; while higher20E titer decrease its transcript level. However, in the adult stage, higher HhEcR transcript level required a relative higher level of20E-induction. It is proved that20E involved in reproductive process of H. hippophaecolus, the activity of reproductive physiology in2-day-old female adult was the highest. These results indicated that fatbody was the mainly tissue responsed the20E level and then futher regulated the transcript activity of20E related genes.4. With the shortening light period and dropping temerature during the late summer, the level of20E, HhSpo, HhPhm, HhShd and HhEcR exhibited seasonal features and the level of pre-overwintering was higher than overwintering period. This paper speculated that the seabuckthorn carpenterworm existed winter hibernation behavior. Their fat bodies provided the necessary energy before winter, and responded to the ecdysone level. Lower20E level coordinated to the lower gene quantity and enzyme activity in fatbody, which regulate the changes of20E titer during development of the insect to further affect the lenghth and life cycle of its larvae. The lenghth of hibernation and life cycle of this species adapted to physiological behavior of wintering period.5. The partial sequence of the seabuckthorn carpenterworm diapause receptor was cloned and the expression profile was measured. Multiple sequence comparation showed the structure of HhDHR display a FXPRL peptide domain of the G-protein-coupled receptors, thus HhDHR may be involved in ecdysteroid biosynthesis of PG.6. This paper described morphological and histological characteristics of the male and female reproductive system of H. hipophaecolus and differences of the male reproductive system between pre-and post-mating. This observation, especially the distribution of sperm buddies, can be used to distinguish mated and unmated males, which may be further applied into the evaluation of the efficiency of sex pheromone traps.In conclusion, fatbody is the main organ to serve as energy storage, responding to the energy signal to regulate metabolic activity, and corresponding to ecdysteroid levels. The HhEcR gene of seabuchthorn carpenterworm fatbody, and the HhSpo, HhPhm, HhShd genes of Holcocerus PG, exhibited seasonal expression characteristics before and after overwintering period as well as coordinated the20E level in seabuchthorn carpenterworm during the entire growth. This regulation characteristic is correlated with the long life span and quiescence of seabuckthorn, which is the adaption to the physiological behavior of overwintering period. |
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