A Study On Characteristics Of Reproductive Dormancy In Overwintering Kallima Inachus

The orange oakleaf butterfly,Kallima inachus(Doyère,1840),famous for its camouflage as dead leaves,is a popular species in butterfly gardens all over the world and a primary cultivated species in southern China in recent years.In natural conditions,it overwinters as adults through its range.This study intends to clarify the nature of its overwintering reproductive dormancy,the sensitive stage(s)to token stimuli for dormancy induction,as well as the natural progress and the environmental factors affecting the maintenance/termination of its overwintering dormancy,by combining field and laboratory observations.The results will provide a theoretical basis for artificially inducing,preventing and terminating the reproductive dormancy of the butterfly,and ultimately provide technical supports for increasing the breeding generations of the butterfly as well as ensuring the supply of adults in winter and spring.1.Determination of the property of the reproductive dormancy in overwintering adults K.inachusThe internal reproductive system of female K.inachus with ages from one-to sixteen-day old were dissected and graded according to the degree of oocyte development.The state of dormancy(i.e.diapause or quiescence)was determined mainly by whether female adults eclosed in the early overwintering period resumed ovarian development under suitable environmental conditions.The internal reproductive organs of female K.inachus comprise a couple of ovaries,a couple of lateral oviducts,a common oviduct,bursa copulatrix gland and spermatheca.There was a significant linear relationship between reproductive development and age at 25?.The ovary development could be divided into four grades.In contrast,after incubation under the same temperature for 12-14 days,most of the females eclosed in early September and all those eclosed in early October only had oocytes without yolk deposition at their age of 24-day,indicating a definite refractory period in these overwintering butterflies.So,The overwintering reproductive dormancy of K.inachus is a typical diapause.The internal reproductive system of males was dissected and numbers of sperm bundle were counted,for observation of the reproductive development progress of the male adults.The internal reproductive system of male K.inachus comprise two testis,two vesicula seminalis,a pair of vas deferens,a pair of ejaculatory duct duplex,an ejaculatory duct simplex and accessory gland.With age increasing,the number of sperm bundles in testis of directly developing male adults increased gradually.In contrast,with age increasing,the number of sperm bundles in testis of overwintering males decreased gradually.2.The natural progress of overwintering dormancy in adult K.inachusThe developmental status of overwintering adults held in conditions similar to the natural environment was inspected at regular intervals to determine the natural period of overwintering dormancy.Overwintering adults were transferred to experimental conditions of 25?(LD 15:9 and RH 70%)at regular intervals to determine when diapause was terminated,as indicated by the resumption of ovarian development.While the majority of female adults in a semi-natural experimental population were reproductively dormant from mid-September to late January,some individuals began ovarian development in early January.Overwintering female adults were in a state of diapause maintenance till the beginning of December,and then gradually entered a period of post-diapause quiescence from late December.From early September to the end of January,the number of sperm bundles decreased gradually.After that,the number of sperm bundles increased from the end of January to February.3.Sensitive stages of overwintering female reproductive diapause to environmental cuesFemale adults were kept in specific temperature and light conditions at their eclosion,and the responsiveness of reproductive development to photoperiod and temperature was observed by dissection.The results showed that the LD 11:13 developmental grades of ovary significantly lower than LD 15:9,the LD 11:13 diapause rates significantly higher than LD 15:9 at 24?.When the photoperiod was fixed to LD 13:11,the 20? developmental levels significantly lower than 24 ?,the 20 ? diapause rates significantly higher than 24 ?,respectively.From newly laid egg to 7-day old adult,experimental insects of different stages(Eggs,1-3 instar larvae,4-5 instar larvae,pupae and 0-7 day old adults)were incubated under long/short daylenths(LD 15:9/LD 11:13,at 24? and RH 70%),or high/low temperature(24?/20?,at LD 13:11 and RH 70%),respectively.The sensitive stages of photoperiod indicates that the early adult stages are sensitive to photoperiod,while the egg and the 1-5 instar larva were not.The ensitive stages of temperature indicates that the early adult stages are sensitive to temperature,while the egg and the 1-5 instar larva were not.The results indicate that the adult diapause of female K.inachus was inducted by low temperature and short photoperiod,and the early adult stages were sensitive to both photoperiod and important for the induction of diapause.4.Effects of environmental factors on the maintenance/termination of reproductive dormancy in overwintering adults of K.inachusOverwintering female adults were exposed to low temperature of 3? for different chilling periods(10/20 d),and were then transferred to different temperature-photoperiod combinations(20?/25?,LD 11:13/LD 15:9)for 10 days,respectively.Finally,they were transferred to common simulated summer condition of 25?,LD 15:9 for 10 days.In experimental group subjected to chilling for 10 days,the developmental levels of ovary were no significant difference in different treatment of temperature-photoperiod combination,and diapause maintenance rates were no significant difference,at the time of dissection.In experimental group subjected to chilling for 20 days,the developmental levels of ovary and diapause maintenance rates were significant difference in different treatment of temperature-photoperiod combination,at the time of dissection.The results indicate that low temperature stimulation for 20 days had a significant effect on the diapause termination of female adults of K.inachus,and that diapause termination was promoted by higher temperature and longer photoperiod,while diapause was maintained by low temperature and short photoperiod.The effect of temperature stimulation was time dependent.