| Phytoseiid mites(Acari: Phytoseiidae)are effective natural enemies and important biological control agents for spider mites.In field conditions,both Phytoseiid mites and spider mites seldom live in fulfillment of all their biotic and abiotic requirements.Rather,they have to face a wide range of environmental stresses that can reduce their fitness,e.g.,insecticides or acaricides,heat or cold stress,drought stress,as well as food supply shortages.Previous study indicated that biological control for spider mites using Phytoseiid predators are frequently being disrupted by high temperatures.However,phytophagous spider mites,e.g.,Tetranychus urticae,Panonychus citri and T.viennensis,are much more thermally tolerant than carnivorous natural enemies,and some species are reported to be betterly adaptive to high temperatures and have a high potential to become serious pests in their warming globe.High temperature and dry weather conditions promote most phytophagous mites abundance,but demote Phytoseiid mite abundance,which cause a direct biological control disruption and failure.Although supplied with plentiful alternative preys,commercial Phytoseiid mites population often remain at relatively low densities by coincidence to hot and dry filed conditions.Moreover,the frequency and magnitude of extreme hot events are predicted to increase with the intensifying global warming.In the middle and lower reaches of the Yangtze River and the southwest and south China,the daily average temperature above 30°C from June to August may last for several days or even tens of days(e.g.,the maximum daily temperature exceeding 39°C lasting for a continuous 9 days in Chongqing in August 2016,and the maximum daily temperature exceeding 35°C last for a continuous 16 days).This resulted in an important research topic in biological control,which is difficult to be solved adequately with the currently commercially available predatory mites and challenges to the current IPM strategies decision makers.High temperature is considered as an important selective agent causing intraspecific variations.Potential adaptations that organisms respond to extreme conditions may concern behavioral and physiological strategies,such as by avoiding stress,changing in growth and reproduction,or expressing of heat shock proteins and inducing enhanced antioxidants.These behavioral or physiological changes that enable them to increase their resistance after pre-exposure to a certain stressful condition are usually referred to as acclimation and/or hardening.However,these adaptations in natural enemies are likely to be inadequate to counter the speed and magnitude of stressful conditions.In this dissertation,we hypothesized that the scenario where biological controls of spider mites using Phytoseiid predators are being frequently disrupted by high temperatures could be ameliorated via a phenotypic plasticity(heat acclimation and heat hardening),which underlies a considerable intraspecific variability for screening a high temperature adapted strain within a certain Phytoseiid predator.We considered a polyphagous predatory mite,Neoseiulus barkeri Hughes as an ideal species for such a screening due to their widely distributions and applications in China,as well as a well-known thermal plasticity in its life history traits.Besides,the commercial available N.barkeri are reared and produced usually under the most appropriate temperature(nearly 25°C).However,their biological control efficiency against pest mites often shows a serious shrink due to its poor adaptability in field conditions.As a result,the stress resistance of this kind of predatory mite is needed to be further strengthened.Therefore,an acclimatory response of N.barkeri via long-term heat acclimations and frequent heat hardenings was carried out in this study.Then thermal susceptibility was measured by using the method and methodology of bio-ecology,physiology and molecular to explain high temperature adaptive mechanism.The results provide a theoretical and practical basis for IPM under climate change.The main results are as follows:1 Heat stress effects on life history traits of Neoseiulus barkeri1.1 Screening for high temperature adapted strainAt the present,the most commercial available phytoseiid mites are raised and produced under the optimum environmental conditions.The stock culture of N.barkeri used in this study was maintained at a constant temperature of 25°C,and it was automatically regarded as the conventional strain(CS)or coincidently as commercial strain(CS),due to either of them was kept at a constant temperature of 25°C.In order to obtain a high temperature adapted strain(HTAS),we initiated the high temperature acclimatory response with approximately 10,000 individuals from its CS colony.Thermal adaptation to high temperature contained two main processes,including short-term heat exposures(heat hardening)and longer-term exposures(heat acclimation).In April 2012,CS mites were firstly heat hardened at 45°C for 2 hours in a programmable temperature controller.The surviving mites were maintained at a heat acclimation condition of 35 ± 1°C and 75 ± 5 % RH in another controller.When the population of N.barkeri reached to the peak value(about 15–25 days after the last heat hardening),the next heat hardening process was performed followed by putting them back to acclimation condition again.HTAS colony had been heat acclimated for 110 generations(450 days)at 35°C and heat hardened at least for 25 times before our experiments.1.2 High temperature susceptibilityAfter long-term heat acclimation and frequent heat hardening,the thermotolerance of N.barkeri to short-term high temperature stress was significantly enhanced.Our results showed that eggs were protected by egg shell and had a powerful thermotolerance.However,it showed a higher mortality when adult females were exposed to heat stress,depending on heat stress magnitude expressed as temperatures and exposure durations.Overall,adult females from HTAS colony showed a stronger and more extraordinary thermotolerance than adult females from CS colony did when taking heat mortality into considerations.At 45°C,the half lethal time(LT50)for CS adults was only 1.9 h,while it was up to 15.2 h for HTAS ones.At the same time,solar exposure at a temperature ranged from 32°C(10:00)to 38.5°C(18:00)in midsummer also caused high mortality in CS adult females.These intraspecific variations on thermal susceptibility suggested that thermotolerance of ectothermic animals could be enhanced by the so-called acclimatory response.HTAS N.barkeri could be considered as a candidate biological control agent and have a potential possibility to solve the current and urgent issue appeared in IPM.1.3 Survival and reproduction under heat acclimation conditionOur results showed that Neoseiulus barkeri had a highly acclimatory response to thermal plasticity.CS N.barkeri could grow and develop safely at a high temperature upon 35°C,whereas its fitness was absolutely lower than that of at 25°C.When CS N.barkeri were maintained at 35°C,an accelerated growth rate,reduced total fecundity and shortened average life span were observed;it was also should be noted that mortality was significantly increased in CS immature stages.HTAS N.barkeri showed an accelerated growth and development rate when maintained both at 25°C and 35°C,respectively.However,the total fecundity and average life span was not different when both strain were maintained at 25°C,which indicated that HTAS N.barkeri might perform a potential reproduction.However,significant lower fecundity was observed in HTAS rather than CS when both strains were maintained at 35°C,which suggested that a trade-off might be existed between reproduction and survival when taking adult female heat mortality into consideration.High temperature acclimatory response might also lead to a 21 % reduced female ratio in HTAS N.barkeri.As a result,N.barkeri might be successfully adapted to high temperature mainly by a bioecological modulation,such as reducing reproduction level to exchange for living opportunity.1.4 Heat stress effects on survival and reproductionBased on the results high temperature susceptibility of adult females,a short-term high temperature stress event(exposed at 38°C and 42°C for 2 h and 4 h,respectively,25°C served as control)was simulated in the laboratory.Adult females(1-2 days after last molting)from CS or HTAS colony were subjected to high temperature stress(Temperature × Duration),and their life history traits were then evaluated at 25 ± 1°C,RH 70–80 % and a photoperiod of L:D = 14:10 h.Adult females were allowed multiple copulations with even-aged adult males to obtain the maximum fecundity.Moreover,Phytoseiid mites must mate and copulate in order to lay eggs.Therefore,adult male fecundity is a critical process in N.barkeri populations.Then,adult females and males from CS colony were used to investigate how a single heat shock event effect on their copulation,longevity,fecundity and egg hatchability through parental effects(adult females were allowed a single copulation only).The results showed that CS N.barkeri were more susceptive to heat stress than HTAS ones owing to their high mortality,prolonged pre-oviposition period,reduced fecundity and longevity.However,when exposed at 42°C,the mean longevity of adult females in HTAS was significantly longer than it was in CS.When adult female heat stressed only(42♀/25♂),or a control adult female mated with a heat stressed adult male(25♀/42♂),or both adult male and female were heat stressed(42♀/42♂),the pre-oviposition period was prolonged,and the oviposition period was shortened and the fecundity was reduced.A sex-specific effect of short term heat exposure on mating behavior was further observed in males where copulation duration of the stressed individuals were somewhat prolonged.In addition,a trade-off between survival and reproduction was observed in heat stressed adult females.However,heat stress had no effect on immediate mortality,pre-copulation period,post-oviposition period,male longevity and egg hatchability of the progeny generations.Our results confirmed that heat stress had a detrimental effect on reproduction,particularly by delaying the onset of oviposition and reducing reproductive output and thereby influencing the population dynamics of N.barkeri.1.5 Heat stress effects on functional responseTo evaluate how heat stress affects the functional response of adult females of N.barkeri preying on nymph T.urticae,we explored the hypothesis at different stressful conditions.The predators were preparedly and firstly heat stressed for 2 h and 4 h at 38°C or 42°C and starved up to 24 h at 25°C,70–80 % RH.Then,the number of prey consumed per predator was recorded after 24 h exposure.The results showed that the adult females of N.barkeri exhibited a type II functional response to nymph T.urticae whether the predators had been pre-exposed to heat stress or not.The functional response of CS N.barkeri was somewhat affected by heat stress,whereas HTAS ones showed differently.Specifically,the number of the nymph T.urticae consumed(24 h)by the heat exposed adult females of N.barkeri were decreased,and the attack rate(a)was reduced,and the handling time(Th)was prolonged,respectively.The value of a/Th was also decreased in response to heat shock.It should be concluded that the decreased consumption on preys might be responsible to prolonged pre-oviposition period in CS N.barkeri.However,these parameters were not changed correlatively to the heat stress intensity.On the contrary,the functional response of HTAS N.barkeri on nymph T.urticae was not affected by heat stress,which indicated that the predators are less vulnerable by high temperature exposure.Heat stress had a negative effect on the appetite of N.barkeri,but the anorexia effect occurred in short term manner and gradually returned to normal level with the extension of recovery time at room temperature of 25°C.2 Heat stress effects on antioxidant responseThermal stress could disrupt the redox balance of cells and cause oxidative stress by the increased reactive oxygen species(ROS)in many organisms.In this study,we examined the time-related effect of the relatively low(0°C and 5°C)or high(38°C and 41°C)temperatures on the activities of antioxidant enzymes including superoxide dismutase(SOD),catalase(CAT),peroxidases(POD),and glutathione S-transferase(GSTs),and the total antioxidant capacity(T-AOC)of the predatory mite N.barkeri.The malondialdehyde(MDA)concentration,as a marker of lipid peroxidation in organisms,was also measured both in CS and HTAS N.barkeri under thermal stress conditions.The results showed that the activities of SOD,CAT,GSTs and T-AOC from HTAS N.barkeri were significantly lower at control conditions than that was observed from CS ones,as well as lower level of lipid peroxidation in HTAS,suggesting that the antioxidant enzymes of N.barkeri might involve in the process of acclimatory response.The activities of SOD,CAT,POD and GSTs were significantly enhanced either in CS or HTAS N.barkeri after exposure to heat stress,suggesting that these antioxidant enzymes might participate in the intracellular antioxidant process by a synergistic manner.However,MDA concentration was accumulated in CS N.barkeri cells with the increased heat stress intensity and magnitude(e.g.,the maximum of MDA concentration was observed at 42°C for 4 h),which suggested that the antioxidant enzymes system had a limited ROS scavenging ability,resulting in a higher level of lipid peroxidation or even caused individual death.Cold stress could induce the up-regulated activities in four antioxidant enzymes.MDA concentrations in CS N.barkeri remained at control levels,suggesting these enzymes could scavenge ROS timely during cold stress.However,the accumulated MDA was observed in HTAS N.barkeri when exposed at 0°C,which indicated that HTAS mites might be more susceptible to cold than CS ones.Our results suggested that N.barkeri might choose to reduce the energy consumption during heat acclimatory response and deal with the short-term temperature stress by cooperating with various antioxidant enzymes.3 Transcriptomic analyses associated with thermotoleranceIn order to obtain a N.barkeri comprehensive transcriptome,the c DNA library was constructed using the mixture of RNA extracted from four developmental stages and generated by Illumina Hi Seq?2000.As a result,a total of 42,917,214 and 43,537,230 clean reads were generated from CS and HTAS,respectively,and further assembled into 52,776 Unigenes.There were 36,951 Unigenes successfully annotated,with an annotation rate of 70 %,in the case of nr,Swiss-Prot,KEGG,COG and GO databases,respectively.The homology analysis of the Unigenes annotated in nr suggested that 52.9 % of N.barkeri Unigenes had the highest similarity to sequences from Metaseialus occidentalis.The HTAS N.barkeri displayed a particular divergent transcriptome profile from CS N.barkeri.As many as 10506 Unigenes expression changed significantly,with 6,338 and 4,168 Unigenes up-and down-regulated,respectively,indicating that the gene expression of N.barkeri in the high temperature environment has an adaptive change.4 HSPs m RNA expression patternsFive Unigenes including CL1558.Contig1_All,Unigene13225_All,CL4607.Contig1_All,Unigene8871_All,and Unigene861_All were identified as heat shock proteins gene by Blastn sequence comparison,candidating for Hsp90,Hsp75,Hsp70,Hsp60 and Hsp40,respectively.The RT-q PCR primers of the 5 HSPs genes were designed using Primer 3(http://bioinfo.ut.ee/primer3/),and Beta actin was used as an internal control using the 2-??Ct method.Firstly,we analyzed the 5 HSPs genes m RNA expression patterns of N.barkeri at different developmental stages.The results showed that the relative expression of a certain HSPs m RNA was significantly different among developmental stages.The relative expression levels of Hsp90 m RNA was measured higher at egg and adult stages than others in CS,whereas a high expression level was found at nymph and adult stages in HTAS.The Hsp75 m RNA was relatively high at adult females for CS and nymphs for HTAS,respectively;Hsp60 m RNA was low at nymphs and adult stages for CS,and egg and adult males for HTAS,respectively.Hsp90 m RNA,Hsp75 m RNA and Hsp60 m RNA were differentially expressed in HTAS in comparison to CS,suggesting that these HSPs might play important roles in the acclimatory response.As the most important molecular chaperone proteins,Hsp70 m RNA and Hsp40 m RNA showed a significantly up-regulated with the increased developmental stages both in CS and HTAS N.barkeri.Secondly,we analyzed the expression patterns of five HSPs at different developmental stages of CS and HTAS N.barkeri after a short-term heat exposure at 38 or 42°C for 1 hour.The results showed that 1 h heat exposure could result in a significantly up-regulated HSPs m RNA expression in N.barkeri and that the expression patterns were highly depended on stress temperature.For example,the relative expression level of Hsp70 m RNA at egg,larva,nymph,adult male and adult female CS N.barkeri was up-regulated as much as 1258.6-fold,2443.9-fold,269.4-fold,135.2-fold and 42.9-fold,respectively when exposed at 42°C for 1 hour;similar results were also found in HTAS ones,with Hsp70 m RNA up-regulated over 1739.2-fold,1478.9-fold,215.5-fold,159.9-fold and 45.9-fold at egg,larva,nymph,adult male and female,respectively.Thirdly,we also analyzed the duration effects of heat stress(exposed at 42°C for 1,2,3 and 4 hour)on the HSPs m RNA expression profiles in adult female of N.barkeri.The relative expression levels of Hsp70 m RNA and Hsp40 m RNA in CS N.barkeri were significantly up-regulated and then gradually returned to control levels or slightly higher than control levels with the stress time prolonged.It should be noted that adult female from HTAS showed a relatively higher levels of Hsp70 m RNA and Hsp40 m RNA expressions than CS did,indicating the two HSPs might be participated in protection from longer heat exposure.The results also showed that the expression profiles for Hsp90 m RNA and Hsp60 m RNA in N.barkeri had an obvious time effects after heat exposure.We concluded that heat shock proteins,such as Hsp90,Hsp70,Hsp60,and Hsp40 of N.barkeri might play important roles in the process of acclimatory response.In summary,the present dissertation was carried out based on the current issue that the biological control of Tetranychidae mites using Phytoseiid mites is occasionally affected by high temperature fluctuations,aiming to enhance the thermotolerance of N.barkeri.At bio-ecology level,HTAS N.barkeri was confirmed more thermally tolerant to high temperature than CS ones when taking their hatchability and adult female immediate heat mortality,survival,total fecundity,longevity and consumption on prey into consideration.In addition,heat stress reduced fecundity in adult males in CS N.barkeri.At physiological level,heat stress could break the redox balance and induce the up-regulated antioxidant enzymes activities both in CS and HTAS adult female of N.barkeri.We also found that the antioxidant response might involve in acclimatory responses in HTAS.At the molecular level,results showed that HTAS N.barkeri displayed a particular divergent transcriptome profile which provided an important tool to investigate its adaptive mechanism to high temperatures.Heat shock proteins might play a vital role in heat shock and high temperature acclimatory response,respectively.The dissertation suggested heat acclimation and heat hardening could enhance the thermotolerance of N.barkeri and therefore obtained a high temperature adapted strain(HTAS),which provides a considerable biological agent for spider mites management in our warming globe. |
PDF Full Text Request