The Sensitive Period And Regulatory Mechanisms For The Shifting Of Migrant Into Resident In The Oriental Armyworm, Mythimna Separata (Walker) (Lepidoptera: Noctuidae)

Migration is the major behavioral strategy for a migratory insect species to adapt the variation of environment, the major causes for the largest number of individual and species in the animal kingdom, and for the frequent outbreaks of migrant pest in agro-ecosystem. A migrant population usually consists of parts of migrant and resident, depending on the environmental conditions that the population underwent. It has long been known that there is a critical or sensitive period when the migrant and resident are determined during the development of ontogeny. This period, was however known to present only in the exopterygota, likes orthopterans, homopterans, and hemipterans, while that in endopterygota, such as lepidopterans and dipterans has not been reported so far. The sensitive period when the migrant was alternated into resident in the oriental armyworm, Mythimna separata (Walker) (Lepidoptera: Noctuidae) was firstly revealed and determined by using ecological, physiological and molecular methods in this paper. The environmental factors and the functions they played in the shifting of migrant into resident were also determined. The shifting of a migrant into a resident was manifested as the short pre-oviposition period, the rapid development of ovary and histolysis of flight muscle and decrease of flight potentials, and all these phenomena were under the control of juvenile hormones (JH) titre, which was synthesized and released by the corpora allata (CA). The Mytse-AT peptide known to regulate JH synthesis was cloned and determined. The Mytse-AT encoding Manse- allatotropin (AT) and the complete coding region sequence was also confirmed. The level of AT mRNA expression coincided with the level of JH titre during the first 5 days of adult life and therefore provided evidences to support the hypothesis that a Manse-Iike-AT was the major regulator for JH biosynthesis in M. separata. The recombinant protein of Mytse-AT obtained was shown to have the function to short the preoviposition period (POP) of a migrant. All of these results provided insights and approaches to understand the evolution and regulation of insect migratory behavior. The major progresses obtained in this dissertation were summarized as follows.The POP was used as a standard to judge a migrant or resident because adult with longer POP usually had a great migratory capacity and there was no apparent morphological variation between the migrants and residents, especially in M. separata. The migrants and residents induced in the laboratory were starved for only 24 hours in the first to sixth days of the adult life. The POP and precalling periods of the migrants starved at the first 24 hours of the adult life was significantly curtailed in comparison with that of the controls while those starved at the second day and thereafter were not able to exhibit such a phenomenon. Furthermore, the life time fecundity of the migrants starved at the first day was also significantly increased while those treated in the later period was decreased. These results demonstrated that a sensitive stage was really presented after the final eclosion and first 24-h in the adult life was thecritical period for the alternation of a migrant into a resident On the other hand, the residents treated by the same environmental factor at the same periods did not show any significant changes in both of POP and life time fecundity, suggesting that resident lacked such a period to shift into a migrant during the adult life. The significances of these results were that the migratory behavior developed in the immature period could be changed by environmental factors that the adult encountered during the first 24 hours post emergence. The life history strategy or the adaptive significances for a migrant was much more flexible than a resident for which lacked any period or chance to alternate its life history strategy even in the extreme environmental conditions.Other two environmental stimulants were also discovered to induce the migrant shifted into the resident in addition to depletion of supplementary nutrition. They were low temperature and illumination or photoperiods, respectively. The POP of migrants was significantly curtailed when they encountered these two factors at first 24 hours of the adult life and the life time fecundity was significantly increased. POP of the migrants encountered the same environmental stimulants in the later developmental periods of the moths did not curtail while the life time fecundity kept unchanged or decreased, depending on the developmental period when these stimulants were encountered. However, the most important thing was that the environmental stimulants, including starvation encountered at the sensitive stage must be stronger or last longer enough to exert their effects. The depletion of supplementary nutrition, for example must be exceeded 6 hours, the illumination must be 24 hours and low temperatures must be at 5 'C for at least 12 hours. These results suggested that the migrants were very sensitive to these stimulants in the critical period and that low temperature, starvation and illumination could induce transform of migrants to resident. However, the same environmental stimulant did not show any effect on the POP when they were applied in the periods beyond the 24 h of the adult life. On the other hand, the same environmental stimulants did not show any significant influence on the residents at any stages of the adult life, except the decrease of life time fecundity. All of these result indicated again, that the adaptability of the migrants to environment was much greater than the residents. The simple reason was that migrant might give up migration and began reproduction at the native habitat when the environmental condition did not allow their migration while the resident could only reproduce in habitat where they were born. All of these discoveries provided a new insight to understand the adaptive significances of migration.To understand the mechanisms of the environmental stimulants acted on the critical period of the migrants, the performance of ovarian development, flight muscle histolysis, flight capacity and JH titre at different developmental stages after the migrants were treated by depletion of supplementary of nutrition at day-1 of the adult life. Ovarian development grade of the treated moths was significantly greater than that of the controls after two days and thereafter of the treatment. Dry weight of the thoracic dorsal-longitudinal muscle in the starved female moths was also significantly lower than that in the control after 120 h of starvation indicating the histolysis of flight muscle was much faster in the treated moth. As a consequence of that, the treated moths exhibited a lower flight potential in comparison withthe control, although the significant difference occurred only since 96 h after treatment. There was also obviously difference in JH titre between the control and treated moths. The levels of JHI titre were not significant greater in the treated moth at 120 h but were significant greater at 144 h after emergence. Level of JHII titre in the treated moth was significantly greater than that in the control since 24 h after the treatment. It was believed that the JHII was likely the major regulator for the ovarian and flight potential development because it was synchronously coincided with the variation of these parameters. Thus, the mechanisms underlying the alternation of migrants into residents in the critical period seemed to be that the corpora allata (CA) was activated during the critical period by the effective stimulants, which resulted in acceleration of the JH synthesis and release. This conclusion was believed so because the level of JH had long been known as the major endocrine regulator for the oogenesis- flight syndrome in the M. separata.In order to seek out the factor regulating the level of JH in the critical period of a migrant, the AT gene of M. separata (herein named as Mytse-AT) known to regulate the JH level was cloned out by using the PCR technique and females brain as template. The accession numbers of Mytse-AT in the GenBank was DQ208707. The Mytse-AT potentially encoded a 136 amino acids' precursor, with calculated molecular weight of 15.385 kDa. The mature Mytse-AT peptide was located between residues 39 and 52, which was completely identical to the sequence of purified Manse-AT peptide and to the prediction of M. sexta AT cDNA. The basic organization of the Mytse-AT precursor was similar to the 135 amino acid precursor of M. unipuncta and similarity was around 97%. Because the Manse-AT peptide was known to be an important gene to regulate JH synthesize in all the lepidopterons, a hypothesis was then proposed that the AT did not express at the critical period of a migrant, and CA was not activated. The Mytse-AT was expressed, and CA was activated and JH was synthesized and released after the migrant received the environmental stimulation at the critical period. The high level of JH titre induced the transformation of migrant into resident. However, further study should be carried out to improve the underlying mechanism because the variation of JH titre in the life of a resident adult had not been able to be investigated in this paper.In order to examine if the level of JH titre was regulated by the Mytse-AT, level of the Mytse-AT expression was investigated by semi-quantity RT-PCR and real time PCR techniques. The results obtained showed that the relationship between AT expression level and JH titre was highly coincided although it was slightly different as the techniques used varied. The level of Mytse-AT expression in the treated moth was obviously greater than that of the control at the subsequent days after the treatment, as determined by the semi-quantity RT-PCR, which was coincided with the variation of JH titre in the treated and untreated moths. The expression of Mytse-AT determined by the real time PCR technique was also coincided with the JH titre in both the treated and untreated moth except at day 5 of the adult life, when the expression of the Mytse-AT in the treated moth was lower than that of the control. These results suggested that JH titre, especially JH II was principally regulated by the level of Mytse-AT gene. Thus, the mechanisms underlying the transformation of a migrant into a resident were that the increasinglevel of Mytse-AT expression activated the CA and enhanced JH synthesis and releasing.Although great progress had been made in the study of AT, few researches were conducted to understand the function of AT gene. In order to confirm if the Mytse-AT gene obtained was active properly, a bacterial expression vector of pET/AT was therefore, constructed. And result obtained showed that it was successfully expressed in E. coli. The recombinant proteins with HIS-tag were also purified. The recombinant protein was demonstrated to be able to activate M. separata CA to synthesize JH because the POP of the migrant treated by Mytse-AT bacterial expression product at the critical period was significantly curtailed while that of the migrants treated by pET21-b bacterial expression product alone, and/or acetone was not significantly shorted in comparison with the control. This achievement indicated that the regulation of migratory behavior in the M. separata had entered a molecular period.