Study On Artificial Rearing Of Cydia Pomonella (L.) And The Mechanism In Response To Thermal Stress

Cydia pomonella (L.) (Lepidoptera:Tortricidae) is an important cosmopolitan quarantine pest of fruits and nuts, whose larvae would result in the decreasing of the fruits quality, the descending of the output and caused tremendous economic loses. C. pomonella originated from the south central Europe, and it endangered the areas except eastern Asia which produce apple and pear all over the world as the spreading of variety of apple. The first damage location of the pest in China was Kuerle in Xinjiang region in 1950s and then sprawling into the inland, at present it had spread over the Dunhuang, Jiuquan and Zhangye region. There are also some invasion reports about the Inner Mongolia Autonomous and the Ningxia Region. All of the damages indicated that the C. pomonella is sprawling into the inland through the natural parclose of the Hexi Corridor, which seriously threatened the fruit-producting and exporting, making the situation becomes more and more serious.Most of the previous research focused on the monitoring and the integrated pest control, so the knowledge of the occurrence rule, ecological adaptability and the spreading mechanism of C. pomonella were relative poor.This study taken C. pomonella as our target, based on the rearing technology of maintaining a laboratory population of C. pomonella on a large scale with the improved artificial diets. According to the ecological adaptability would facilitate serious invasion. The key studies were that, (1) Effects of different kinds of artificial diets on the growth and fecundity of the C. pomonella laboratory population; (2) Flight capacity and the starvation tolerance of the C. pomonella population; (3) Thermal adaptation and molecular mechanism of its thermostability of the laboratory population. This study is aim to enrich the invasive mechanism of this important pest, providing biology basis for precisely estimating the spreading ability and the suitable survival extent of C. pomonella and laying down the reasonable quarantine and preventive Steps. The main results are as follows:1. Effects of different kinds of artificial diets on the growth and fecundity of the C. pomonella laboratory populationReferring to the international artificial diets, we obtained three kinds of improved artificial diets, wheat germ apple powder diet, wheat germ diet and the pinto bean diet. Based on the artificial diet, a healthy laboratory population of the C. pomonella was established. The important life parameters of this pest, such as weight of pupa, fecundity of female and hatching rate, still maintained at a proper level after 14th generation on artificial diets, especially for the eggs laid per female and the hatching rate was kept at the range 124.2-143.2 and 78.0-88.5% respectively. These parameters were consistent with the field capacity. Among the three kinds of artificial diets, the wheat germ apple powder diet could produce the population with highest quality. The intrinsic rate of increase (rm), finite rate of increase (λ), net reproductive rate (R0) and mean generation time (T) of the C. pomonella laboratory population were 0.085d-1,1.09 d-1,43 offspring and 44.6 d when fed on the Pinto bean artificial diet kept at 27℃。2. Flight capacity and the starvation tolerance of the C. pomonella populationFlight capacity of C. pomonella adult was measured by flight mill, the results indicated that they had strong flying ability. The flight distance of female and male was 12.16km and 10.58km respectively in 16h testing process. The proportion of the strong flying capacity individual which exceeds 10km was 58.3% and 37.5%. The result of the starvation endurance of different instar larvae of C. pomonella showed that the longest time for 1th-5th larvae were 44.7h、73.4h、141.2h、194.8 h and 203.6 h respectively. The endurance time was significant prolonged as the age increased, the starvation endurance time of 4th and 5th larvae were significant higher than the early age larvae. Lacking of nutrition did not significantly influence the longevity of female and fecundity. So we come to an conclusion both the adult and larvae had strong transfering and expanding ability.3. Thermal Adaptation of the C. pomonella laboratory populationThe tolerant threshold of eggs and various instars were measured under extreme temperature, the result showed that the mortality of eggs was significant increased as the temperature raised when treated at 48℃and 50℃for 2,5 and 10 min respectively. Eggs were killed 100% when exposed 50℃for over 5min. lth-5th larvae were exposed at 50℃and 52℃for 2,5 and 10 min, respectively. The result indicated that the 3th larvae were the most sensitive stage and the 5th were the most resistant stage under extreme high temperatures. lth-4th larvae were killed 100% when exposed 50℃,10 min and 52℃,5 min, but for 5th larvae, their survival rate still maintained at 25.0% and 11.1% respectively. The 5th larvae were killed 100% only happened when exposed 52℃over 10 min. The result demonstrated that the 5th is the most resistant stage under extreme high temperatures. The effects of thermal stress on survival and fecundity of C. pomonella 5th revealed that there was no significant difference of survival rate when 5th instar larvae exposed to 32-44℃for 3 h, the pupae recovery rate and the longevity of the female were not affected when exposed at 32～38℃for 3 h. But the longevity and fecundity were significant reduced when treated temperature higher than 38℃. Maybe there had a tradeoff between the high 5th survival rate and the adult fecundity.4. Molecular mechanism of thermostability for the C. pomonella laboratory population1) Cloning and sequence analysis of the heat shock proteinBased on the conservative traits of heat shock gene families, using degenerate primer for homology-based cloning, combinated with the technique of RACE, we obtained one hsp90 gene and two hsp70 genes from 5th female larvae which exposed to 35℃for 3h. They were assigned name Cphsp90, Cphsp70-1 and Cphsp70-2 respectively. And then analysised the Open Reading Frame and predicted molecular weight of each full-lenth cDNA. the final Motif "MEEVD" or "EEVD" indicated the three hsps were the Cytoplasmic heat shock protein. They shared 96%,90% and 87% with other organism respectively. For CpHsp90, the relationship was relative near to Carposina sasakii, for CpHsp70-1 and CpHsp70-2, near to Plutella xylostella.2) The expression profiles of Cphsp90 and Cphsp70 genes from C.pomonellaThe expression profiles of Cphsp90 and Cphsp70 genes from C. pomonella were analyzed by using the RT-qPCR technique with actin gene as reference gene under both low-temperature (4-25℃) and high-temperature(32-44℃) stress for 1 h. The result as follows:(1) Cphsp90 and Cphsp70-1 mRNA up-regulated when exposed 4℃during a series low-temperature strees, but the Cphsp70-2 mRNA down-regulated, it did not response significantly to the low-temperature stress. (2) Cphsp90, Cphsp70-1 and Cphsp70-2 mRNA up-regulated when exposed to 32-44℃, the relative expression level was 12,50,80 times compared with 27℃control. The result indicated that the three hsp genes had important role in deal with heat stress.3) Development stages and tissues specific expression profiles of hsps(1) The result of the relative expression level in eggs, lth-5th larvae, pupae and adults which kept at 27℃constant time indicated that the Cphsp70-1 and Cphsp70-2 mRNA in the 3th-4th larvae significant lower than other life stages, almost did not exist. So we consider the two genes was developmental regulated, which demonstrated highly in eggs and early age larvae, little in 3th-4th larvae. This was coincide with the conclusion that the 3th larvae was the most sensitive stage under extreme high temperatures. Three kinds of hsp genes were responsed when all of the stages exposed under 35℃for 1 h. eggs,1-2th,5th and the adults were up-regulated.(2) The result of the relative expression level in different tissues which kept at 27℃constant temperature indicated that there was significant difference between hemolymph, midgut, fatbody and the curtile. Cphsp90 was highest in midgut and fat, Cphsp70-1 and Cphsp70-2 were highest in epidermis. After 35℃heat treatment, all of the three hsp genes were significantly up-regulated in epidermis, and singnificant higher than fat and midgut, which proved that the epidermis was the most active organization of the heat shock gene mRNA. Maybe this is the hot mechanism of epidermis during the pest adaptability in the changing environment.4) C. pomonella diapaused involved hsps and the relationship with the heat resistanceThe thermostability of 5th diapaused larvae were stronger than non-diapaused ones. We take the overwinter diapause 5th larvae and the artificial induced diapause larvae as materials, make a parallel research on the coordinatingly expressing level of heat shock gene in the process of diapause. The result indicated that Cphsp70-1 mRNA were involved in the overwinter diapause process, and significantly up-regulated, for artificial induced diapause larvae, was Cphsp90. However, the Cphsp70-2 was down-regulated in the both. When the diapause larvae were exposed to 32-44℃temperature for 1 h, all of the three kinds of hsps were significant up-regulated to protect the organism. Maybe the hsp involved the diapause process is the main factor for the diapaused larvae possessing the strong heat resistant to high temperatures.5) Theromotolerance plasticity mechanism for the larvaeHeat tolerance of C.pomonella were affected by various conditions which they undergone. Theromotolerance plasticity would be caused by the fluctuate temperatures and preheat treatments. We take the common reared larvae, overwinter larvae and the artificial induced diapaused larvae as materials, make a research on the relationship between the hsps expression and the theromotolerance plasticity. The three kind of larvae which undergo different temperature fluctuation, exposed to higher temperatures after preheat treatment under 35℃for 3 h, take the non-preheated larvae as control. The result indicated that the Cphsp90 mRNA were rised when exposed 40-50℃after preheat treatment, and need higher stress temperature to induced the ultimate expression, and the expression pattern were the same in the three kinds of larvae which undergo different conditions. So we make a consideration that the theromotolerance plasticity of the 5th larvae related closely with the Cphsp90 expression profile, and not be affected by the experience of temperature fluctuation, its adjusting pattern is steady. Compared with Cphsp90, the expression pattern of Cphsp70 was affected by diapaused, because Cphsp70-1 and Cphsp70-2 were both inhibited when exposed to higher temperatures after preheat treatment in the common reared larvae, up-regulated in the same treated diapaused larvae. So we make another consideration that the two Cphsp70 up-regulated involved in the diapause when under high-temperature stress maybe another important factor for the higher theromotolerance with diapause larvae.Contribution and novelty of this paper:(1) This study established an laboratory population of C. pomonella with the artificial rearing technique on improved artificial diets for the first time in China. And chieved multigenerational consecutive maintaining of the laboratory population.(2) First identification the flying capability and the starvation tolerance of Chinese C. pomonella laboratory population.(3) Identified the tolerant high-temperature threshold of eggs and various instars, and the effect of thermal stress on survival and fecundity of the laboratory population.(4) Bsed on the phenomenon that the temperature fluctuations might lead to theromotolerance plasticity, explored the molecular mechanisms of C.pomonella response to thermal stress, and the role in the theromotolerance plasticity.