| The oriental fruit moth, Grapholita molesta (Busck), is a key pest of stone fruit, its feeding habits is miscellaneous, and it has a broad range of host plant. In recent years, due to the agricultural structure adjusted and the climate changed, resulting in most large-scale infestations of this pest had occurred throughout the growing season in many regions every year. In some regions, this pest had become a major pest according to the populations and harmful degree, it has caused serious economic losses. In the Northwest of China, this pest overwintered as mature diapausing larvae for about five months under natural conditions. Whether the diapausing larvae can successfully overwinter, affects the population dynamics of this species in the following year. Studies on the diapause ecology of oriental fruit moth have been documented. However, very little work has been devoted to investigate the physiological and biochemical mechanisms of diapause in this species. To improve understanding of the diapause characteristics and its surroundings impact factor in this species, further to know the regulatory effect of diapause on the population dynamics of this pest, provide novel targets for pest forecasting and integrated control. In this paper, the ecological, physiological adaptation mechanisms of diapause of this pest were conducted in-depth in experimental conditions. The main results were summarized as follows.1. The photoperiod response type, critical photoperiod, sensitive stage of diapause induction and the effect of temperature on diapause induction in oriental fruit moth were determined in the laboratory. In ecological light cycle, this pest was a typical short-day diapause type. At20℃, when the photoperiod was8L:16D,10L:14D and12L:12D, the diapause rate>95%, the critical photoperiod was13.75L:10.25D. At24℃, when the photoperiod was10L:14D and12L:12D, diapause rate>95%, the critical photoperiod was13.68L:10.32D. During the larval stage, the age in different day of larvae were treatmented under12L:12D and16L:8D light respectively, all results showed a certain degree of sensitivity to diapause inducting photoperiod, although there was no obvious critical point in time, but still the age in5-8th day of larvae was more sensitive to diapause inducing photoperiod. The larvae continoued in the short day length was longer, the rate of diapause was higher. Temperature was not a key influential factor in diapause induction, but could adjust the effect of short photoperiod on diapause induction. At the mediam temperature of10-25℃, low temperature could promote diapause induction. The low temperature of5℃and the high temperature of30℃, to some extent, inhibited diapause induction of short photoperiod. Temperature played an important role only in the dark period.2. Photoperiodic clock mechanism of diapause induction in oriental fruit moh was investigated. The photoperiodic response under24h light-dark cycles showed that the critical night length was10.25h at20℃and10.32h at24℃, high incidences of diapause were at intermediate scotophases of12-16h. Under non24h light-dark cycles, the response curves of diapause induction were significantly different in different photophase, but the length of12h in scotophase all resulted in the highest rate of diapause. The length of scotophase played an essential role in diapause induction, but the length of photophase could adjust the diapause inducting effect of scotophase. In night interruption experiments showed that the percentage of diapause was significantly lower in the scotophase of10L:14D,11L:13D and13L:11D which were interrupted by1h light pulse than that of12L:12D, the most highly photo-sensitive position occurred in the middle of the dark period, and interruption effects had a relationship with photoperiod. Nanda-Hamner and Bunsow experiments showed that the percentage of diapause were all significantly lower and the diapause induction curve did not show rhythmic fluctuations within24h in this species. These results suggested photoperiodic clock in this pest showed an hourglass-liker response model or a rapidly damping oscillator model.3. At4℃,10℃and24℃, diapause termination of oriental fruit moth were systematically investigated. Diapause larvae could be terminated by exposing them to low temperature of4℃and10℃for120days, it seemed to be more conducive to complete diapause development at the medium-low temperature of10℃compared to the low temperature of4℃. Diapause larvae could not be terminated at24℃until day120. The result indicated that low temperature had a significantly effect on diapause termination. Diapause larvae only in low temperature, experienced a period could be terminated.4. The anthrone colorimetry, acetylacetone colorimetry and coomassie brilliant blue G-250dying were used to analyze the dynamic metabolic processes of total sugars, trehalose, glycogen, glycerol and protein in diapausing larvae oriental fruit moth. At low temperatures, glycogen content decreased significantly, its metabolic rate was different under different temperature, and utilization rate was higher at4℃than10℃. The change of total sugar content was fairly similar with glycogen. Trehalose content increased significantly during diapause development at4℃and10℃, as well as at24℃and maintained at a higher level. Glycerol level was significantly increased at4℃and showed three peaks, while at10℃and 24℃, glycerol content did not significantly accumulate, it showed that only reached at determinate low temperature, the synthesis of glycerol could be induced. Glycerol is mainly cryoprotectants of oriental fruit moth during diapause period. Protein content increased during diapause development at4℃、10℃and24℃, changed insignificantly at4℃and10℃and significantly at24℃. During diapause period, the changes of the content of those test chemical substances were significantly affected by diapause development and temperature and could reflect the cold-resistance of overwintering larvae of oriental fruit moth.5. The activities of several metabolic enzymes including trehalase (TRE), sorbitol dehydrogenase (SDH), hexokinase (HK), pyruvate kinase (PK) and alkaline phosphatase (ALP) in diapausing larvae oriental fruit moth were measured using3,5-dinitrosalicylic acid colorimetry, dual reagent method and metabolic enzyme activity testing kits. The activities of these metabolic enzymes were significantly different during diapause development at low temperatures. Initially TRE activity significantly increased, then gradually decreased with the prolongation of the time of diapause. SDH activity changed more gently and was no larger fluctuation. HK, PK and ALP activities significantly reduced during the early phase, and then fluctuated at a lower level during the late phase. During diapause period, diapause intensity and temperature had significantly affected on these metabolic enzymes. The metabolic rate remained at low level through the regulation of metabolic enzymes could improve the ability of cold hardiness of oriental fruit moth.6. The content of hydrogen peroxide (H2O2) and the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) in diapausing larvae of oriental fruit moth were determined by using the protective enzyme testing kits. At low temperatures, H2O2content was significantly increased, the activities of SOD and CAT decreased during the early phase of diapause, then significantly consistent increased. POD activities decreased in the early stage of diapause, and maintained at low level. During diapause period, H2O2content and protective enzymes activities were significantly affected by temperature and diapause intensity, and had a relationship with cold hardiness.7. The dynamic of polyamine content and its metabolic enzyme activities of oriental fruit moth were examined by high performance liquid chromatography, UV colorimetry and iodine-starch colorimetry. At low temperature, putrescine (Put) content slightly decreased in the early period of diapause, then rapidly rised, spermidine (Spd) content increased and maintained high level during the whole diapause development, Put and Spd content was significantly higher at4℃than that of at10℃, spermine (Spm) content decreased in the early phase of diapause, then significantly increased in the late phase of diapause. The activities of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) increased significantly, ODC activities were higher than SAMDC, indicating the biosynthetic pathway of ornithine is the main way of polyamine metabolism in oriental fruit moth during diapause development. The activities of polyamine oxidase (PAO) also increased during diapause development, and were significantly differences at4℃, but no significantly differences at10℃and24℃. During diapause period, polyamine metabolism was significantly affected by diapause development and temperature, and the polyamine accumulation could help to improve diapause larvae of hardiness at low temperature. |
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