| The tea leafhopper Empoasca onukii Matsuda is one of important insect pest in tea production and causes a dramatic decrease in tea yield and quality, which is an outstanding pest problem to be solved urgently in the world. Both adults and nymphs actively move around to feed on fresh tender leaves causing characteristic symptoms referred to as “hopperburn”. The average tea loss due to the damage by this pest is about 10~15% each year. This insect can also cause a dramatic decrease in tea quality and a loss of over 50% quantity in some temperate years. In mid-south of China, E. onukii are active from mid-March to early December, and peak numbers occur in July and October. Populations sharply decline in later November with decreasing temperature. In the summer, this insect usually spends its life on deciduous trees and in the winter on conifers or evergreens adjacent to tea fields. It is now apparent that there is no physiological diapause in this pest and the optimal temperature range for survival, development and reproduction of E. onukii is 20~25?C. Therefore, E. onukii is frequently exposed to high or low temperature stress in nature. The temperature is normally above 20?C during this period in China, occasionally with a temperature higher than 40?C or lower than 0?C. Under the goblal warming, extreme-high and low temperature is expected to increase in frequency and intensity, therefore, how can E. onukii adapt the temperature stress is crucial to their existence and development, but research on this aspect is rare now.Comprehensive information about the changes in the level of Hsp genes from E. onukii in the response to temperature stress is not available yet. To explore changes of E. onukii to thermal and cold stress conditions at the molecular level, here in this paper, we devoted to investigating the thermaltolerance of E. onukii and their response to high and low temperature, and to exploring the the expression profiles of heat shock protein on heat resistant of E. onukii under the short-term high and low temperature, in order to indicate the influence of temperature on this insect pest and the molecular mechanism on temperature adaptability, meanwhile provide a reasonable measure for prevention and control.1. The developmental duration and fecundity of E. onukii were studied and the life tables were constructed at five different temperatures(15, 19, 23, 27 and 30?C) in the laboratory. The results showed that the developmental rate of E. onukii increased with the increment of temperature at 15~30?C and the relationship between them could be simulated by the Logistic model. The developmental durations of nymph, pre-oviposition period, egg period and generation time were 5.54~18.09, 4.02~6.86, 5.54~14.68 and 15.10~39.63 days at the experimental temperatures, respectively. The threshold temperatures of nymph, pre-oviposition period and egg stage were 36.85, 7.86 and 11.06?C with effective accumulated temperatures of 136.59, 74.98 and 93.54 day-degree, respectively. The effective accumulated temperature of the whole generation was 305.33 day-degree. The life of the female adult longer than male at the experimental temperatures, were 6.48~24.10 and 5.38~19.39 days, respectively. Finite increase rate(0.14, 0.15 and 0.16), Generations survival rate(90%, 89.60% and 87.20%) and Net reproductive rate(18.42, 17.46 and 15.90) were highest at 19~27?C at different temperatures. It follows that the optimum temperature for the development and fecundity of E. onukii was 19~27?C.2. This study was conducted to explore the effect of temperature on the fitness of E. onukii adult in thirteen(-10,-5, 0, 5, 10, 15, 20, 25, 30, 35, 38 and 41?C) temperature treatments. The result showed that the survival rates of E. onukii adults were at the similar level at 5~35?C, both males and females. The survival rates of males were gradual declining starting at 38?C, was significantly different from other treatments, but females undifferentiated at 38?C. As the temperature rose(>38?C) or fell(<5?C), the survival rates declined significantly. A large number of E. onukii was unable to survive for long time above the 44?C or under-5?C treatment points, and the survival rates of females were higher than males. Appropriate temperature stress can prolong the life span of adult, 0?C stress is remarkable. And the life span of adult was shortening significantly under high temperature stresses. The eggs of every female' was decreased under both high and low temperature stress.3. This study was conducted to explore the effect of temperature on the adaptability of E. onukii Matsuda adult in thirteen(-10,-5, 0, 5, 10, 15, 20, 25, 30, 35, 38 and 41?C) temperature treatments. The activities of superoxide dismutase(SOD), copper zinc superoxide dismutase(Cn Zn-SOD), peroxidase(POD) and catalase(CAT), the total antioxidant capacities(T-AOC) and the content of total proteins were measured when the adults had been treated for 1 hour in each temperature group. The results showed that there was no distinction of activities of protective enzymes, T-AOC and total protein content when the adults were treated between 5?C and 35?C and no distinction of activities of Cu Zn-SOD in all treatments, the values fell between 0.96 U/mg and 1.05 U/mg. When the temperature was lower 5?C or higher 35?C, the protective enzymes activities were decreasing or increasing, respectively. The highest antioxidize activity and total protein content appeared at 41?C, with 2.64 U/mg for SOD, 0.68 U/mg for POD, 1.58 U/mg for CAT, 0.67 U/mg for T-AOC and 178.33 ?g/m L for total protein content, respectively. The adults were more sensitive in low temperature, with lower indexes than in high temperature. This tendency indicated that the protective enzymes activities, T-AOC and total protein content were closely related with temperature. The death in extreme temperature may be due to the damage of protective enzymes.4. Two heat shock protein genes were cloned. We cloned two heat shock prootein genes, named Eohsp90(Gen Bank accession number: KF730250) and Eohsp70(Gen Bank accession number: KF730249). The deduced amino acid sequence from Eohsp90 contains 5 typical motifs of the hsp90 family and Eohsp70 contains 3 typical motifs of hsp70 family. The open reading frames of Eohsp90 and Eohsp70 were 2 172 bp and 2 016 bp in length, respectively. Their deduced amino acid sequences of Eohsp90 and Eohsp70 showed high homology with other Hemiptera species.5. The expression patterns of Eohsp90 and Eohsp70 under high temperatures were investigated in our studies. The results showed that the relative expression levels of both Eohsp90 and Eohsp70 in E. onukii adults were up-regulated as the temperature rises or falls over time, except in the-5?C or 44?C temperature groups. Moreover, the expression level in the temperature elevated groups was higher than that of the lower temperature groups. In addition, the Eohsp70 generally demonstrated a higher transcriptional level than Eohsp90, and both genes had a higher expression profile in female adults compared to the males. The expression profiles indicated that Eohsp90 and Eohsp70 may play important roles in E. onukii adult responses to ecologically relevant environmental temperature threat.Our studies imply that E. onukii has the great thermaltolerance, which may be the main reason for the existence on wide range areas and survival in tea plantation occurrence on cold winter days and outbreak on hot summer days. |
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