| Neoseiulus barkeri Hughes,one of the phytoseiid mites(Acari: Phytoseiidae)who were faced with various stress,e.g.,high temperature,low humidity,UV-B,insecticide and acaricide,when were released into the field.Among these stressors,high temperature and low humidity always occur simultaneously.The relative humidity in leaf surface layer decreases with the increase of temperature,especially when the plant is subjected to drought stress or the surrounding environment is windy.The relative humidity during the hottest hours in a summer day often dropped below 50% in southwest of China,e.g.,Chongqing.Compared with pest mites,predatory mites are more sensitive to high temperature and low humidity condition,this led to the outbreak of spider mites.In previous studies,the stress that arthropds confronted with was often evaluated based on single stress,while two stressors usually impact on arthropds in a synergistic way,indicating that the synergistic effect from two stress is usually greater than the sum of effect of a single stressor.Therefore,it is necessary to evaluate the threat from a multiple-stressor perspective.Stress hardening and acclimation has been tested effective in enhance tolerance of predatory mites to stress condition.However,these researches only focused on single-stressor situation,the method to improve tolerance of organisms to the simultaneous two stressors remains largely unknown.To find out how the combined effect of high temperature and desiccation impact on survival of N.barkeri,females were held at different temperature and humidity.We observed 6 h stress at 38 ℃,50% RH is lethal to female mites.To increase the survival time of female mites under heat and desiccation stress,mites were subjected to high temperature,desiccation and high temperature×desiccation hardening and then the mortality rate was compared at high temperature and low humidity condition to screen out the effective hardening method.Based on transcriptome analysis,metabolite analysis and gene expression pattern analysis of female mites under hardening condition,we comprehensively analyzed the physiological mechanism and water metabolism of female mites in response to stress hardening.After this,the way to enhance tolerance of female mites to high temperature×desiccation stress by sugar feeding was explored.The main results are as follows: 1 Survival and water metabolism of N.barkeri female under high temperature and low humidityFemale mites of N.barkeri were held at different temperature and humidity combinations.The half lethal time and the time that 95% of female mites died at certain temperature and humidity combination(TH50,TH95)was calculated.Considering that water vapor pressure deficit(VPD)is better showing the “drying power” of air than RH dose,RH was converted to VPD(mbar).With the increase of temperature and VPD,the survival time of female mites decreased significantly.We also observed significant interactive effect of high temperature and VPD on survival time,which indicated that high temperature and VPD impacted on female mite in a synergistic way.Water loss rate of female mites significantly increased with temperature and VPD,and it correlated significantly with survival time,indicating water loss is one of the main reasons that caused death of female mites under heat and desiccation stress.Female mites had maximum survival time at 25 ℃,90% RH and 100% RH,where mites had the TH50 of 403.6 h and 328.6 h and TH95 of 604.6 h and 512.6 h respectively.And female mites losed less than 4.5% of total weight within 26 h,indicating mite was able to keep its water balance at 25 ℃,90% RH and 100% RH and this condition is suitable for its living.At 38 ℃,TH50 was 43.9 h and 97.42 h separately,and mites gained weight and absorbed water vapor from air at the first 5 h.However,mite lost more than 20% of its total weight after 24 h at 90% RH and 39 h at 100% RH,indicating that female mites can not maintain water balance at high temperature for a long term.When temperature increased from 41 ℃ to 43 ℃ and 45 ℃,TH50 and TH95 decreased at a rate quicker than that the WLR increased,which indicated that heat stress decreased survival time of female mites at 43 ℃ and 45 ℃.At 38 ℃,50% RH,the common temperature and humidity in summer in southwest of China,TH50 of female mites was 4.1 h,and TH95 was 6.1 h.2 Stress hardening on mortality of N.barkeri female under heat×desiccation stressIn order to reduce the mortality rate of female mites under heat×desiccation stress,female mites were hardened at high temperature,desiccation and heat×desiccation stress seperately.After hardening and recovery,mites were stressed by heat×desiccation(38 ℃,50% RH and 41℃,50% RH)to compare the hardening-induced effect on mortality rate.The results showed that hardening at high temperature did not reduce the mortality of female mites under heat×desiccation stress.On the contrary,hardening at high temperature increased mortality of female mites under heat×desiccation significantly,with the increase of hardening duration and temperature,the mortality of female mites under heat×desiccation stress increased.All hardening conditions causing water loss significantly reduced mortality under heat×desiccation stress.Water loss amount(WLA),rather than WLR or temperature during hardening,significantly correlated with mortality under heat×desiccation stress,indicating that the amount of water loss during hardening significantly affected the tolerance of female mites to heat×desiccation stress.Hardening at 25 ℃,0% RH and 25 ℃,50% RH was more effective in improveing survival than other conditions.Female mites that hardened at 25 ℃,0% RH have higher body weight and lower WLR under heat×desiccation stress,indicating that the reduced mortality under heat×desiccation stress was mainly caused by reduceing WLR and increasing bulk water content of female mites.3 Physiological mechanism of N.barkeri female in response to desiccation hardening 3.1 Transcriptomic analyses during desiccation hardeningTranscriptome analysis was performed on female mites that were hardened at 25 ℃,0% RH for 0 h,4 h,and 6 h.After sequencing and filtering,a total of 3 × 107 to 8 × 107 clean reads were got in a sample,GC content was between 50%-52%,and the Q30 was greater than 94%.After assembly,a total of 30621 genes were obtained,in which 17150 genes were annotated.In trend analysis and KEGG pathway enrichment analysis,most up-regulated genes were enriched in the catabolism of saccharides,fatty acids and other macromolecular substances,gluconeogenesis pathways and oxidative phosphorylation;Down-regulated genes were enriched in signal transduction,glycolysis,polysaccharide synthesis,fatty acid chain elongation and secondary metabolite synthesis.The above results suggested accelerated catabolism of macromolecules into small molecules,e.g.,glucose,throught gluconeogenesis pathway,and reduced synthesis of macromolecules,reduced metabolic rate,and the accumulation of small molecules,e.g.,glucose.The accumulation of small molecules(e.g.,sugars)and reduced metabolic rate all contribute to reduced water loss of arthropods faced with dehydration stress,which may partly explain the hardening-induced effect on female mites under heat×desiccation stress.3.2 Metabolite profile of N.barkeri female during desiccation hardeningAfter GC-MS analysis,sycllo-inositol,glucose and myo-inositol were the main tested component in female mite body.Among these three components,sycllo-inositol accounted for 65.91%,glucose accounted for 19.12%,and myo-inositol accounted for 14.96%.Concentration dynamic of the above three carbohydrates in female mites was analyzed during desiccation hardening,recovery,and heat×desiccation stress.The glucose content of female mites ranges from 2.63×10-4 to 5.85×10-4(μg/μg).During desiccation hardening,glucose content decreased gradually;After recovery,glucose content began to increase and was significantly higher than that in control;Female mites who have experienced desiccation hardening had higher glucose content under heat×desiccation stress.Thus the glucose concentration was in accord with mortality change under heat×desiccation stress.There observed no significant difference in concentration of scyllo-inositol during desiccation hardening,recovery and heat×desiccation stress between hardened mites and non-hardened ones.Although there observed increased concentration of myo-inositol in desiccation hardening and recovery,the difference was not significant.At heat×desiccation stress,myo-inositol concentration in hardened female mites was lower than that in non-hardened ones,which did not comply with the lower mortality rate of hardened female mites under heat×desiccation stress.The above results suggested that the accumulation of glucose is one of the important reasons for the decreased WLR and mortality of female mites under heat×desiccation stress.3.3 Glucose feeding and expression patterns of glycometabolism related genesfemale mites fed with 20% glucose(w/v)had lower WLR under heat×desiccation stress,which was consistent with the results of desiccation hardening.During desiccation hardening,the gene of glucose-6-phosphatase(Nb G6Pase)in gluconeogenesis pathway was significantly up-regulated expressed,while gene in the pentose phosphate pathway(Glucose-6-phosphate 1-dehydrogenase,Nb G6PD)and glycolytic pathway(Hexokinase,Nb HK1)significantly down-regulated expressed.During recovery,Nb G6 Pase,Nb G6 PD,Nb HK1 and Nb HK2 all up-regulated expressed.We speculated that the increased glucose concentration was mainly caused by gluconeogenesis.When stressed at heat×desiccation(38 ℃,50% RH)for 2 h,the expression levels of Nb G6 Pase,Nb G6 PD,Nb HK1 and Nb HK2 in female mites who were desiccation hardened were lower than those were non-hardened;However,when stressed for 3 h,the expression levels of these genes in hardened female mites were up-regulated compared to 2 h,while the expression levels of those genes in non-acclimated female mites were down-regulated.This suggested that female mites who have experienced desiccation hardening had higher tolerance to heat×desiccation stress.3.4 Effect of desiccation hardening on respiratory rateThe respiratory water loss is positively correlated with metabolic rate and CO2 emission in insects and other arthropods.By comparing respiratory rate between desiccation-hardened female mites and non-hardened mites at heat×desiccation stress(38 ℃,50% RH),we found that there was no significant difference in respiratory rate between hardened and non-hardened female mites.We speculated that the hardening-induced effect on mortality under heat×desiccation stress was not caused by different respiratory water loss.4 Effect of dietary glucose and trehalose on mortality of N.barkeri female at heat×desiccation stress 4.1 Different effect of dietary glucose and trehalose on mortality of female mites under heat×desiccation stressDietary glucose and trehalose including the concentration of 10%,20%,40% reduced the mortality of female mites under heat×desiccation stress.With the increase of dietary trehalose concentration,the mortality rate of female mites decreased;While with the increase of dietary glucose concentration,the mortality rate of female mites increased.We selected the concentration(20% glucose and 40% trehalose)that had lowest mortality rate under heat×desiccation stress to feed female mites.We found that female mites fed with 40% trehalose had higher mortality rates under heat×desiccation stress than those fed with 20% glucose.However,female mites fed with 40% trehalose had higher water content,indicating that the reduced mortality rate was partly caused by containing more bulk water in female mites fed with 40% trehalose.According to metabolites concentration of female mites after feeding,higher concentration of glucose and inositol were both found in female mite fed with 20% glucose and 40% trehalose,with no significant concentration difference of glucose and inositol between mites fed with 20% glucose and 40% trehalose.However,trehalose was only found in female mites fed with 40% trehalose,with the concentration as high as 1.901×10-3 μg/μg,suggesting that this part of trehalose had no obvious effect enhancing tolerance of female mites to heat×desiccation stress.And feeding with 20% glucose was more effective in enhancing the tolerance of female mites to heat×desiccation stress than feeding with 40% trehalose.4.2 Effect of sugar feeding after desiccation hardening on mortality of female mites under heat×desiccation stressIn order to further reduce the mortality of female mites under heat×desiccation stress,we fed female mites with 20% glucose or 40% trehalose after desiccation hardening.We found that female mites who were fed with 20% glucose or 40% trehalose after desiccation hardening had lowest mortality rate under heat×desiccation stress than female mites that were only fed with sugar or only desiccation hardened.Body weight after feeding was compared,and no significant difference was found between the hardened and non-hardened female mites after 20% glucose feeding,while significantly higher body weight was observed in hardened female mites compared with nonhardened ones after 40% trehalose feeding.This suggested distinct mechanism of dietary glucose and trehalose to enhance tolerance of female mites to heat×desiccation stress after desiccation hardening by absorbing more glucose and more water content respectively.After fed with 20% glucose or 40% trehalose after desiccation hardening,the survival rate of female mites under heat×desiccation stress decreased with time,close to the survival rate of non-acclimated female mites after 48 h.In summary,the combined stress of high temperature and low humidity will greatly shorten the survival time of female mites and accelerate their WLR.Desiccation hardening is effective in improving survival of female mites under heat×desiccation stress by activating gluconeogenesis and depressing glycolysis,polysaccharide synthesis,fatty acid chain elongation,signal transduction and secondary metabolite synthesis,leading to accumulating of glucose.The increased glucose concentration led to lower water loss rate and higher survival rate of female mites under heat×desiccation stress.Dietary glucose was more effective in increasing survival rate of female mites under heat×desiccation stress than dietary trehalose.And the survival rate of female mites can be furtherly increased by feeding glucose or trehalose after desiccation hardening.This study demonstrated the survival of female mites under heat×desiccation stress,and the stress hardening methods and mechanism to increase survival of female mites to heat×desiccation stress,which will provides us theoretical clues in controlling the spider mites under heat and desiccant condition using predatory mites,e.g.,N.barkeri. |
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