| The frequency of sudden extreme weather increases due to global industrialization.How to adapt to cold temperatures and sudden cold waves in winter is a great challenge to the insects in the temperate and subtropical regions.The level of insect cold hardiness is an important prerequisite for the existence and development of insect population in poor low temperature environment.Sericinus montelus is a high-value ornamental butterfly species but is very rare and endangered.S.montelus overwinters with diapause pupae.Whether the cold hardiness is related to diapause and the specific mechanism is still not clear.Therefore,in order to reveal the physiological characteristics of the mechanism of cold tolerance of S.montelus overwintering pupae,diapause and non-diapause S.montelus were raised in the laboratory and outdoors in this study.The intelligent detector was used to detect the supercooling points of diapause-destined pupae and non-diapause pupae.The water content,energy substances,cryoprotectants and enzyme activities related to cold hardiness of diapause-destined pupae and non-diapause pupae were compared and analyzed by biochemical substance content test and High Performance Liquid Chromatography(HPLC).The respiratory pattern and metabolic rate of diapausedestined pupae and non-diapause pupae were measured and compared by portable respiration measurement system.At the same time,the above indicators of pupae in different seasons in the wild were detected.Finally,the effects of rapid coldhardening on cold hardiness and cryoprotectants were analyzed.The main results are as follows:1.Supercooling pointThe results show that the supercooling point of diapause-destined pupae is significantly lower than that of non-diapause pupae,and the supercooling point of overwintering pupae is significantly lower than that of summer pupae.The supercooling point of diapause-destined pupae and overwintering pupae is-26℃ and-24℃,respectively,which is far lower than the minimum temperature in winter in Hunan.The survival emergence rate of diapause-destined pupae was 20%after cold exposure at-24℃ for 2 h.The supercooling point was highly correlated with the lower lethal temperature.Therefore,reducing the supercooling point is one of the main cold-resistance strategies for overwintering pupae in diapause.It is speculated that Sericinus montelus is a freeze-avoidance insect.2.Water contentThere was no significant difference in free water and bound water content of non-diapause pupae at each age.The free water content of diapause pupae decreased and the bound water content increased with the increase of pupae age.Except for 1 day old,the free water content of diapause pupae was significantly lower than that of non-diapause pupae,and the bound water content was significantly higher than that of non-diapause pupae.At the 1st,4th and 8th day after pupation,there was no significant difference in total water content between diapause-destined pupae and non-diapause pupae.The 12-h dehydration rate of diapause pupae was significantly lower than that of non-diapause pupae,and the 12-h dehydration rate of diapausedestined pupae decreased significantly with the increase of pupa age.These results suggested that diapause-destined pupae of S.montelus might improve its cold hardiness by reducing free water content.3.Energy substancesThe protein content of diapause-destined pupae and non-diapause pupae decreased after pupation,but there was no significant difference between diapausedestined pupae and non-diapause pupae.There was no significant change in the protein content of overwintering pupae.The contents of total sugars,lipids and glycogen in non-diapause pupae decreased rapidly before adult emergence.The total sugar content of diapause-destined pupae increased gradually after pupation,and the changes of pupae glycogen and lipid content were opposite to the changes of total sugar content.The contents of lipid,glycogen and total sugar in diapause-destined pupae during diapause maintenance period(8d after pupation)were significantly higher than those in non-diapause pupae.Pupae of diapause are depleted in the early stage of diapause,and the glycogen may be converted to monosaccharides or sugar alcohols,which turn to lipids after 30 days of diapause.These results indicate that diapause-destined pupae can store energy in the form of lipids,glycogen and sugars.and can use this energy during the metabolic inhibition period during diapause.4.Low-molecular-weight cryoprotectantsThe contents of low-molecular-weight cryoprotectants in diapause pupae,nondiapause pupae,summer pupae and winter pupae were determined by HPLC.Eight standard freeze protectants commonly found in insects were selected,and the contents of trehalose,glucose and sorbitol in the insect body were reached the limit of quantization.The results showed that the glucose and sorbitol contents in diapause pupae and overwintering pupae had no significant changes related to cold hardiness.Trehalose content in diapause pupae increased significantly from 7 μg/mg at the beginning of pupation to 16 μg/mg at the 30th day after pupation.The concentration of trehalose in.outdoor pupa increased from 9 μg/mg in summer to 12 μg/mg in November and remained high in winter,but decreased to 4 μg/mg in March next year.The outdoor trehalose and supercooling point of pupae showed opposite trend.Therefore,it is inferred that trehalose is the cryoprotectants of the pupae of the S.montelus.5.Enzyme activity related to cold hardinessThe activities of catalase(CAT),peroxidase(POD),superoxide dismutase(SOD)and ethanol dehydrogenase(ADH)in diapause pupae,non-diapause pupae and outdoor overwintering pupae were compared.The results showed that the activities of SOD and POD had no significant change related to cold hardiness.CAT activity of one-day-old non-diapause pupae was significantly higher than that of diapausedestined pupae,and ADH activity of diapause-destined pupae began to increase 4 days after pupation,and remained at a high level within 30 days.CAT activity of overwintering pupae decreased significantly after overwintering,ADH activity increased significantly during overwintering and decreased significantly in March.These results suggested that CAT and ADH enzymes might be related to the improvement of cold hardiness in overwintering pupae of diapause-destined S.montelus.6.Respiratory pattern and metabolic rateThe respiratory metabolism and respiratory patterns of diapause-destined pupae and non-diapause pupae,summer pupae and overwintering pupae were detected and compared.The results showed that there were at least two respiratory patterns:continuous and discontinuous gas exchange.All diapause-destined pupae,winter pupae,and non-diapause pupae at low temperature(15℃)showed discontinuous gas exchange respiration pattern and low metabolic rate,while most non-diapause pupae,all summer pupae showed continuous gas exchange respiration pattern.The duration of the whole cycle of discontinuous gas exchange,burst period and inter diapausedestined period were extended with the increase of pupae age and the decrease of temperature.7.Effects of rapid cold-hardening on cold hardiness and low-molecular-weight cryoprotectantsNon-diapause pupae were acclimated at 4℃ and diapause-destined pupae were cold acclimated at 0℃ and 4℃ for 2,4 and 8 h.The results showed that acclimation at 0℃ for 2 h could significantly improve the emergence rate of diapause-destined pupae.After 8 days of diapause acclimation at 0℃ for 2 h,8 h,24 h,8 d and 22 d,there was no significant difference in the content of low-molecular-weight cryoprotectants in the pupae compared with the control group.The results indicated that rapid cold-hardening under appropriate conditions could improve the cold hardiness of diapause-destined pupae,and this change was independent of cryoprotectants.The innovation of this study is mainly reflected in the following aspects.It is confirmed that S.montelus is an insect that avoids freezing,and the diapause of the pupae is closely related to the cold tolerance.Variability was found in the respiratory pattern in the pupae of S.montelus.It was found that rapid cold acclimation could signifi cantly improve the cold tolerance of diapause pupae. |
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