Analysis Of The Responses Of Bradysia Odoriphaga To High Temperature Stress And Its Thermal Adapation Mechanism

The Chinese chive root maggot,Bradysia odoriphaga Yang et Zhang?Diptera:Sciaridae?,the specific devastating underground pest in China,damaged various crops,and caused severe losses on Chinese chives.Since the huge population and stronge insecticide resistance,it is diffcult to control effectively,leading to severe safety risks for agricultural products.Its population dynamic showed obvious seasonal variation,with outbreaks in spring and autumn,and population decreases in summer.Previous studies have confirmed that temperature was one import environmental factor affecting the abundance of B.odoriphaga.However,the effects of heat sress on B.odoriphaga and its thermal adaptation had been reported scarecely.Here,we analyzed the effects of high temperature stress on the life parameters of B.odoriphaga to assess its thermatolerance,and investigated its adaptation strategy through heat acclimation,thermal endurance,progeny fitness and host-population.Furtherly,we determined the antioxidant response,Heat shock protein?Hsp?gene expression,and hormone metabolism of B.odoriphaga under heat stress to clarify the thermal adaptation mechanism.Our results provided valuable theoretical basis of the adapatation metabolism against various environmental stress factors,and key technical supports of the application of high temperature on B.odoriphaga control.The main results are as follows:1.High temperature exerted dramatic adverse effects on the development and reproduction of B.odoriphaga,and short-term heat shocks over 36°C caused obvious lethal and sublethal effects.Life-table study under 25³2°C indicated when temperature exceeding30°C,the survival declined,and development delayed,and adults longevity shortened,and fecundity decreased.32°C was the highest temperature at which B.odoriphaga could complete development.Over 36°C,short-term heat shock showed rapid lethal effects.Adults and eggs possessed weaker thermatolerance,and the median lethal time(LT₅₀)at 38°C was between 1.10¹.40 h;4^th instar and pupa possessed stronger thermatolerance,and LT₅₀ were1.83 h and 2.15 h respectively.After heat shock,the life parameters of survival B.odoriphaga were suppressed,and adults suffered more servious negative effects.After 4^th instar exposed at 38°C for 2 h,the larval development times prolonged by 3.63 d,and the fecundity decreased by 23.95%,and the hatching rate declined by 12.56%.After adults exposed at 38°C for 2 h,the fecundity decreased by 75.63%,and the hatching rate declined by 60.97%.2.Our study confirmed that 4^th instar was the most key stage to adapt heat thermal stress.The thermal tolerance was significantly enhanced by heat hardening;4^th instar larva kept alive at sublethal high temperature stress?34°C?.After long-term high temperature treatment,the percentage of female of offspring?F1?increased.The thermatolerance of B.odoriphaga reared on different diets was discrepant.The above adaptation stratages played the key role in spending the hot summer and restoring population in autumn for B.odoriphaga.The thermal tolerance of 4^th instar larvae was significantly enhanced afer heat hardening,and the acquired-thermotolerance increased gradually with the increasing temperature?30³6°C?;At 12 h after heat hardening?34°C and 36°C for 4 h?,the survival rate of 4^th instar larva exposed to 38°C for 1 h was 58.50%and 61.75%,which was higher than the control.Additionally,when these larvae developed to adult stage,the survival rate of female adults exposed to 38°C for 1 h was 49.75%and 51.50%,still higher than the control.However,there was less obvious effect of heat hardening on the thermotolerance of adults.Under sublethal high temperature stress?34°C?,the development of 4^th instar larvae stagnated,and failed to pupate.While,the larva kept alive for a long time,at 20 d and 30 d,the survival rate was 46%and 12%.When temperatures became favorable,the survival larva needed a period of restoring time to resume development.After treatment at 34°C for 20 d and 30 d,the development time before pupation was 9.89 and 10.56 d respectively,and the pupation rate was 80%and 64%.However,the female longevity shortened significantly compared with at 25°C,and fecundity declined by 51.69%and 40.46%.The other stage exerted no long-term adapatation.The female sex-ratio in the contemporary populations?F0?of 4^th instar larva exposed at34°C for 20 d and the constant population at 32°C declined.The sex-ratio?Female:Male?of4^th instar at 34°C for 20 d and constant population at 32°C was 0.76:1 and 0.72:1.However,the female percentage in the first filial generation?F1?was 1.45:1?34°C for 20 d?and 1.47:1?32°C?higher than that at 25°C?1.05:1?.The reproductive characteristic of the parent female under different hot condition indicated that higher temperatures accelerated the parent female skewing toward a high proportion of females with single females or female dominance per progeny,which was 45.7%at 25°C,and 59.3%of 4^th instar treated 20 d at 34°C,and 57.9%at32°C.Additionally,the thermatolerance of the first filial generation was enchanced by high temperatures.The LT₅₀ of 4^th instar larva?F1?at 38°C was 1.91 h?34°C for 20 d?and 1.97 h?32°C?.The life parameters of B.odoriphaga reared on different diets differed obviously.When reared on Chinese chive and oyster mushroom,B.odoriphaga showed better life parameters than on humus and garlic.Comapred with reared on Chinese chives,rearing on garlic and humus,the larval developmet times delayed by 2.48 and 7.48 d,and the larval survial declined by 23.15%and 33.50%,and the fecundity decreased by 27.18%and 48.40%.Furthermore,the thermatolerance of 4^th instar larva reared on 5 kinds of diets differed.Rearing on garlic and humus,4^th instar larva better tolerated heat stress,and the LT₅₀ at 38°C were 1.92 and 1.89 h,which longer than rearing on Chinese chive?1.58 h?and oyster mushroom?1.69 h?.3.Furthermore,we assayed the physiological and molecular responses of B.odoriphaga under heat condition.The results demonstrated improving autioxidant enzymes activities and synthesis of Hsps were the important responses of B.odoriphaga to heat stress.Short-term heat shock led to oxidative damage on adults.To maintain homeostasis and prevent ROS damage,antioxidative enzymes activeties strengthened.CAT and SOD activities exerted the strongest at lower temperature?36°C?;While,POD and GSTS showed the strongest at higher temperature?38°C?.Furtherly,under sublethal heat stress?34°C?,the activeties of antioxidative enzymes maintained higher level.For 20 d,the activeties of CAT,POD,SOD and GSTs were 1.71,2.08,2.14 and 1.44 times higher than that at 25°C.Additionally,the difference of antioxidative enzymes activeties of 4^th instar reared on different diets was in accord with the thermatolerance.4^th instar reared on garlic and humus possessed higher antioxidative enzymes activeties than that fed on Chinese chives and oyster mushroom.Short-term heat shock induced the expression levels of hsp20,hsp40,hsp60,hsp70 and hsp90 up-regulated.The expression levels hsp70 and hsp90 up regulated more dramatically than the others,being the most important Hsps involved in heat stress.After heat shock at38°C for 1 h,the hsp70 and hsp90 expression was up-regulated by 19.92-and 15.29-fold.Furtherly,we found the hsp70 and hsp90 expression levels of 4^th instar after heat hardening up regulated,but decreased as the times going on.Additionally,under long-term sublethal heat stress,the expression levels hsp70 and hsp90 maintained high-level expression.At 20 d,the expression levels was 10.03?hsp70?and 7.74?hsp90?times higher than that at 25°C.4.We determined the contant of juvenile hormone and 20-hydroxyecdysone of 4^th instar larva under long-term sublethal heat stress.The results indicated that the high level of juvenile hormone and low level of 20-hydroxyecdysone regulated the development of 4^th instar larva stagnation collectively.At 34°C for 10 d and 20 d,the juvenile hormone content was 2.61-and 3.28-fold higher;while the 20-hydroxyecdysone content was 0.75-and 0.65-fold lower.The relative expression levels of juvenile hormone synthesis gene?JHAMT-1?kept higher level,while the hydrolysis gene?JHEH?kept lower expression level.5.Closing the Chinese chive greenhouse at high temperature in winter could be a very feasible measure used in the ecology control for B.odoriphaga.On the basis of the theory that adults showed weaker thermatolerance,the control efficacy of the measure of closing the Chinese chive greenhouse at high temperature in winter during the high occurrence period of adults could attain 50⁶0%.During this period,the temperature over 40°C was held for 3⁴ h in every day.Combining with other methods,the control efficacy could be enhanced furtherly.