Effects of short-term heat shock and physiological responses to heat stress in two Bradysia adults, Bradysia odoriphaga and Bradysia difformis

Bradysia odoriphaga and Bradysia difformis are devastating pests of vegetable, ornamental crops and edible mushrooms causing significant losses. Temperature may be an important factor restricting their population abundance in the summer. To determine the effects of short-term heat shock on adults, their survival, longevity and fecundity data were collected, and antioxidant responses and heat shock protein expression levels were examined. Our results indicated that the survival rates of Bradysia adults decreased rapidly after heat shock ≥36 °C, and the longevity and reproductive capacities were significantly inhibited, indicating that short-term heat shock had lethal and sub-lethal effects. Moreover, the lipid peroxidation levels of B. difformis and B. odoriphaga increased dramatically at 36 °C and 38 °C, respectively. Four antioxidant enzymes activities of B. odoriphaga were greater than those of B. difformis at 38 °C. Additionally, hsp70 and hsp90 expression levels significantly increased after heat stress, and higher expression levels of B. difformis and B. odoriphaga were discovered at 36 and 38 °C respectively, indicating their different heat tolerance levels. Overall, short-term heat shock (≥36 °C) caused significantly adverse effects on Bradysia adults, indicating that it could be applied in pest control, and antioxidant system and hsp genes played important roles in their heat tolerance levels.

ROS, which causes oxidative damage 18 . The surplus ROS causes lipid peroxidation (LPO) and disrupts cell membrane fluidity, resulting in cell lesions 18 . The degree of membrane LPO can be determined indirectly by measuring the malondialdehyde (MDA) concentration 19 . To maintain homeostasis and prevent ROS damage, organisms have evolved complex adaptation-related mechanisms for eliminating ROS, including molecular antioxidants and anti-oxidative enzymes 20 . Antioxidative enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and glutathione-S-transferases (GSTs), are the most important components for protecting cells and maintaining homeostasis involved in various stress conditions by scavenging ROS 21,22 . Many studies have measured antioxidant responses under thermal stress conditions as indicators of the important physiological adaptation processes of insects, including Corythucha ciliata 23 , Bactrocera dorsalis 24 and Plutella xylostella 25 .
Inducing Hsp (heat shock protein) gene expression levels is an important physiological adaptation to biotic and abiotic stresses. HSPs act as molecular chaperones that participate in maintaining regular cellular functions and in regulating metabolic activity, thereby protecting cells from oxidative damage 14 . Among the different heat shock proteins, Hsp70 and Hsp90 belong to two major conserved families and are commonly expressed under thermal and other stress conditions. In addition to preventing oxidative damage, they may also interfere with the signaling events that trigger the apoptotic process 26 . In previous studies, hsp90 and hsp70, as stress markers, have played important roles in resisting high-temperature stress and in protecting insects from thermal injury and death 14,27 . The different expression levels of hsp70 and hsp90 correlate positively with the thermotolerance of insect species and populations 27,28 .
Bradysia odoriphaga Yang et Zhang and Bradysia difformis Frey, two main root maggot flies, are devastating pests of liliaceous vegetables, flowers and edible fungi, and they can coexist on the same host plant in protected cultivation or in open fields [29][30][31][32] . Their larvae tend to aggregate to attack and damage roots and corm tissues, resulting in moisture loss and even death 31,33,34 . In Chinese chive fields, the two Bradysia species occur with similar regularities, with outbreaks in the spring, autumn and winter in greenhouses, and population decreases in the summer 35,36 . Temperature was thought to be an important factor affecting their population dynamics during different seasons. The optimum temperature ranges of the two Bradysia flies were 13-28 °C for B. odoriphaga and 10-25 °C for B. difformis, and a temperature over 30 °C had adverse effects on both species. The development threshold temperature (T 0 ) of B. odoriphaga was 6.29-8.7 °C, while 4.0-8.4 °C for B. difformis 32,34 , and we hypothesized B. difformis had a lower optimum temperature range and threshold temperature, indicating greater cold tolerance than B. odoriphaga. Because extreme daytime temperatures can exceed 35 °C for several hours during the summer season in northern China, high temperature was regarded as a critical abiotic factor restricting their occurrence in the summer. However, there are no reports regarding the thermal tolerance levels of the two Bradysia flies against heat stress. Our previous work indicated that the two Bradysia species were sensitive to heat stress, additionally, adults stage being the most sensitive stage to heat shock (unpublished). Other researchers also confirmed that heat shock negatively influences B. odoriphaga 37 , but no research about heat tolerance of B. difformis was reported. To manage root maggot flies efficiently in Chinese chive fields, it is important to clarify the impact of high temperature on the survival and fecundity of these pests, which will aid in predicting their occurrences.
In this study, we demonstrated the lethal and sub-lethal effects of heat shock on two Bradysia adults. The physiological responses to heat stress in the two root maggot flies were then determined, including those of the antioxidant systems and hsp gene expression levels. Our findings provide an important theoretical basis for predicting population dynamics and understanding the potential physiological adaptations to heat stress for two important Bradysia flies.

Results
Lethal effects of heat shock. When the temperature excessed 36 °C, heat shock exerted lethal effects on both Bradysia adults (Table 1 and Fig. 1). As an example, the heat shock at 36 °C for 1 h resulted in B. difformis survival rates of 80% (female) and 84% (male), while B. odoriphaga was not affected. When the temperature increased to 38 °C, the B. odoriphaga survival rates were 53% (female) and 62% (male), while those of B. difformis were 28% (female) and 34% (male), and at 40 °C, no B. difformis survived, while the B. odoriphaga survival rates were 11% (female) and 19% (male).

Discussion
Poikilotherms are usually exposed to various challenges to survival and reproduction in their environments, and temperature is a critical abiotic factor that causes physiological changes in arthropods 24 37 . Thus, this environmental condition is extremely adverse to Bradysia species. Because it absorbs the solar radiation, the ground's surface temperature is greater than the atmospheric temperature, which could aggravate the heat stress. Previous studies confirmed that Bradysia adults, having weak flight capabilities, were mainly active on the ground 32,38 . Thus, the two Bradysia adults were bound to confront thermal stress in the summer, and the thermal stress resulted in rapid death, restricting their abundance. In addition to rapid lethal effects, heat stress also exerted various biological stresses on surviving insects, such as suppressing fecundity and longevity 1,10 . Previous studies confirmed that two Bradysia adults did not oviposit  at once after emergence, and the pre-oviposition period ranged from 1.0 to 1.5 d [32][33][34] , indicating that they were restricted to suffer the heat stress in the daytime before oviposition. Results obtained in this study revealed that heat shocks (36-38 °C for 0-4 h) suppressed the reproductive capacity and longevity of the surviving adults. After exposure to 38 °C for 2 h, few B. difformis survived, and those that did were unable to oviposit and mate. Meanwhile, more B. odoriphaga adults survived, but their fecundity was low. Our findings were partially consistent with the previous reports about other insects. Cheng et al. also found heat shock, except for rapid lethal effects, also resulted in longevity and fecundity suppression of B. odoriphaga adults 37 . Agasicles hygrophila adults  suffered adverse effects after being exposed to 36 and 39 °C for 4.0 h, with the fecundity and offspring hatching rate decreasing 39 . Similarly, the longevity of Helicovrpa armigera was shortened under heat shock (40-46.5 °C) and the fecundity decreased 40 . The effects of heat stress on fecundity and longevity in insects may be a result of direct injuries to reproductive systems or metabolic disorders. However, we could not determine whether the reduction in fecundity resulted from damage to the reproductive systems of both sexes or to only one sex. Heat stress (36-40 °C) exerted significant lethal and sub-lethal effects on two Bradysia adults, but there were significant differences in the heat-tolerance responses between them: B. odoriphaga possessed more heat tolerance than B. difformis indicating that the former maintained a higher survival rate after heat shock exposure and suffered less severe sub-lethal effects. Previous research confirmed that Bemisia tabaci, whose population peaked in summer, did not exhibit significant negative changes after a 1-h heat shock (37-45 °C), while the fecundity of Trialeurodes vaporariorum, whose population peaked under cooler conditions, decreased rapidly 10 . Thermal adaptability limits the distribution and abundance of Culicoides imicola and Culicoides bolitinos. Compared with C. imicola, C. bolitinos has a wider altitude range and has stronger heat-and cold-stress tolerance levels 41 . Moreover, B. dorsalis, a widely distributed species, whose heat tolerance is enhanced by heat hardening at 35 °C, 37 °C, 39 °C and 41 °C, has a greater thermal plasticity than Bactrocera correcta, a narrowly distributed species, whose heat tolerance is only enhanced at 39 °C and 41 °C 42 . Here, the regional distributions of the two Bradysia species in Chinese chive fields were significantly different. B. odoriphaga has a wide distribution in North China, especially in Shandong, Hebei and Beijing, while B. difformis is mainly distributed in the northwest and northeast of China 31,32,36 . We hypothesized that the different responses to heat shock were related to the population dynamics of the two Bradysia species.
Generally, exposure to high-or low-temperature stress may lead to oxidative damage and generate surplus ROS in insects 43,44 . To relieve the adverse oxidative stress, insects increase antioxidant defense to maintain a balance in ROS metabolism 45 . For example, the antioxidant enzyme activities (SOD, POD, CAT and GSTs) of Bactrocera dorsalis 24 , Chilo suppressalis 44 , Antheraea mylitta 46 and Propylaea japonica 47 were induced by heat stress to protect the insects. In this study, after 36 and 38 °C heat shock treatments, the MDA concentration of B. difformis was greater than that of B. odoriphaga, which indicated that B. difformis suffered more oxidative stress. Moreover, the antioxidant enzyme activities (SOD, POD, CAT and GSTs) varied significantly after heat stress, indicating the protection function of antioxidant enzymes. At 36 °C, the POD, CAT and GST activities of B. difformis were greater than those of B. odoriphaga, while all of the tested antioxidant enzyme activities of B. odoriphaga were greater than those of B. difformis at 38 °C. This phenomenon was consistent with that B. odoriphaga possessed a stronger heat tolerance than B. difformis. Furthermore, the POD and GST activities of B. odoriphaga were induced at a higher temperature (38 °C), suggesting that they were stimulated to protect insects by scavenging ROS at a higher heat stress. Meanwhile, the reduction in the SOD activities in both Bradysia adults at 38 °C, compared with the control, suggested that excessive ROS could decrease SOD activity. Thus, the different heat tolerance levels in the two Bradysia species were related to the different responses of antioxidant enzymes to heat stress.
Hsps of insects are involved in physiological responses to various environmental stresses, especially heat and cold stress 15,42 . Previous research confirmed that Hsp70 and Hsp90 were two prominent Hsps that play important roles in thermal stress. Our study also confirmed that in both Bradysia adults the expression levels of hsp70 and hsp90 were induced by heat stress, suggesting that these two Hsps were involved in protecting Bradysia adults from thermal stress. At 36 °C, the relative expression of hsp70 and hsp90 in B. difformis increased more significantly than in B. odoriphaga, while the opposite was true at 38 °C. Previous studies indicated that the temperature for the onset of the induction of hsp gene expression (T on ) and the temperature for the maximal induction of gene expression (T max ) of hsp may be useful indicators to evaluate the thermal tolerance of insects 48,49 . The higher of T on and T max of hsps are, the stronger heat tolerance of insects is, while the lower of Ton and Tmax of hsps are, the stronger cold tolerance of insects is. The T on and T max of five hsps (hsp20, hsp40, hsp60, hsp70 and hsp90) in Liriomyza huidobrensis, which possesses a great heat tolerance, were higher than in Liriomyza sativae, which has a greater cold tolerance 49 . Similarly, Drosophila virilis, the low-latitude species, possesses a greater heat tolerance than Drosophila lummei, the high-latitude species, and the T max of the expression levels of the hsp genes in the former were greater than in the latter 28 . Thus, in the current study, we hypothesized that the T on and T max of hsp70 and hsp90 in B. odoriphaga were close to 38 °C, higher than those in B. difformis, close to 36 °C. Indeed, B. odoriphaga possessed a greater heat-tolerance than B. difformis. Moreover, the synthesis of Hsps and antioxidant enzyme proteins consume biological energy 50 . Thus, the declines in the fecundity and longevity of the Bradysia species may have resulted from a reduction in energy, which was consumed to synthesize stress proteins or supporting enzymatic reactions.
In conclusion, our results confirmed that two Bradysia adults were sensitive to heat shock and that after short-term heat shocks their longevity and fecundity were suppressed. B. odoriphaga possessed the greater heat tolerance, and the difference in the heat tolerance levels between species was related to protective physiological responses, such as antioxidant capacities and hsp expression levels.

Methods
Insect materials. Bradysia odoriphaga Yang and Zhang and Bradysia difformis Frey colonies were originally obtained from a Chinese chive greenhouse field in Tai'an, Shangdong, China, in April 2015. Insect colonies were maintained in the Shandong Provincial Key Laboratory of Applied Microbiology, and reared on Chinese chives for more than 5 generations according to the breeding method 32,34 . Eggs, larvae and pupae were reared in culture dishes (Φ = 9 cm) covered with wet filter paper, and newly emerged adults were placed in pairs in oviposition containers (3-cm diameter × 1.5-cm height). Insect colonies were maintained in growth cabinets at 25 ± 1 °C with 75 ± 5% relative humidity, and a 12:12 h light:dark cycle. Heat shock treatment. The treatment methods refer to the methods described by Huang et al. 9 . Adults (single-sex) that emerged from pupae within a 12-h period were collected in a 10-mL centrifuge tube, and exposed to a water bath at the target temperature (32, 34, 36, 38, 40, 42 and 44 °C) for 0.5, 1, 2 and 4 h. They were allowed to recover at 25 °C for 1 h. The survival number was recorded. The treatment kept at 25 °C was regarded as the control. Each treatment contained 100 individuals for five replicates, and each replicate contained 20 individuals. The median lethal temperatures, L temp 50 values, were calculated according to the logistic regression (1).
Longevity and reproductive capacity. Above lethal experiments indicated that 34 °C was the highest temperature exerted no lethal effects on adults within 4 h, while almost no B. difformis adults survived at 40 °C. Therefore, after the heat shock (34, 36 and 38 °C for 0.5, 1, 2 and 4 h), the surviving adults were collected as the tested insects. Males and females were paired and placed on oviposition plastic and reared at 25 °C. Adults were checked every 12 h, and the numbers of eggs were recorded until all of the adults died. The average longevity, fecundity and female fertility rate were calculated. The treatment kept at 25 °C was regarded as the control. Every treatment contained 60 pairs of adults for three replicates, and each replicate contained 20 pairs. Antioxidant responses and hsp70 and hsp90 expression levels. Heat treatment. Above lethal experiments indicated that there were significant differences in survival rate of two Bradysia adults at 36 and 38 °C for 1 h. The new adults (single-sex) were exposed to a water bath at the target temperature (36 and 38 °C) for 1 h, and then allowed to recover at 25 °C for 1 h. The treatment kept at 25 °C was regarded as the control. All of the surviving adults were flash frozen in liquid nitrogen and stored at −80 °C.
Sample preparation and enzyme activity assay. The treated adults were homogenized in a cold mortar with a pestle in 0.05 M phosphate buffer solution, pH 7.8, containing 0.1 mM ethylenediamine tetraacetic acid and 1% polyvinylpyrrolidone. The crude homogenates were centrifuged at 10,000 g for 15 min at 4 °C. The supernatant was gathered for the determination of antioxidant enzyme activities. Protein concentrations were determined using the Bradford assay. The activities of CAT, POD, SOD and GSTs, and the MDA concentration, were determined by spectrophotometry. All spectrophotometric analyses were conducted in a Shimadzu UV-2450 spectrophotometer (Shimadzu, Arlington, MA, USA). Every treatment contained 240 individuals (single-sex) for three replicates, and each replicate contained 80 individuals.
CAT activity was calculated by measuring the consumption of H 2 O 2 at 240 nm for 2 min. The amount (μmol) of H 2 O 2 decomposition per min per mg protein was defined as one unit of CAT activity. The result was expressed as U mg −1 protein.
POD activity was assayed using the guaiacol oxidation method at 470 nm. One unit of POD activity was defined as the amount that catalyzes 1 μmol substrate reaction per minute per mg protein. The result was expressed as U mg −1 protein.
SOD activity was measured based on the inhibition of the nitro blue tetrazolium photochemical reaction at 550 nm. One unit of SOD activity was defined as the amount of enzyme that caused a 50% inhibition of the nitro blue tetrazolium reduction. The result was expressed as U mg −1 protein.
GSTs activity was determined using 1-chloro-2,4-dinitrobenzene and reduced glutathione as the substrate. The change in absorbance was measured continuously for 4 min at 340 nm. Changes in absorbance per min were converted into mmol 1-chloro-2,4-dinitrobenzene conjugated/min/mg protein using a molar extinction coefficient of 9.6 mM −1 cm −1 . The result was expressed as mmol min −1 mg −1 protein.
The LPO was determined indirectly by measuring the amount of MDA formed by reacting with thiobarbituric acid to give a red species having a maximum absorption at 532 nm at 37 °C 25 . The MDA concentration was expressed as nmol of MDA produced per mg protein.
Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). Total RNAs were extracted using an RNApure Tissue Kit (DNase I) (ComWin Biotech, Beijing, China). cDNA was synthesized using the SYBR1 PrimeScript RT-qPCR Kit II (Takara Biotechnology, Dalian, China). hsp70 and hsp90 mRNA levels were measured using RT-qPCR. RT-qPCR reactions were performed using the Bio-rad CFX96 Real-Time PCR System (BioRad Laboratories, Hercules City, CA, USA) with SYBR-Green detection. The average threshold cycle (Ct) was calculated per sample. The relative expression levels were calculated with the 2 −ΔΔCT method. The relative level of each hsp was defined as the increase (in folds) compared with the amount of β-actin. RT-qPCR primers and the list of accession numbers are provided ( Table 3). The process of how to design these primers was supplied in the Supplementary section (Table 1S, Figs 1S and 2S). Each gene was analyzed in triplicate in each of three biologically independent treatments. Every treatment contained 150 individuals (single-sex) for three replicates, and each replicate contained 50 individuals. Data analysis. In the logistic regression analysis (Eq. 1), the survival rates of these two Bradyisa adults after heat shock were regarded as the dependent variable (y), while the treated temperatures were regarded as the independent variables, and ×0 indicates the L temp 50 value. We tested the variables for homogeneity of group variances using Levene's test and normality using the Kolmogorov-Smirnov test prior to statistical analysis. For analysing the difference among different treatments, the survival rate, longevity, fecundity, egg hatching rate and female spawning rate of B. odoriphaga or B. difformis at each heat treatment were regarded as the dependent variable, while the treatment were regarded as the independent variables in one-way ANOVA followed by Tukey's HSD multiple comparisons. For analysing the difference between two species, the survival rate, longevity (single-sex), fecundity, egg hatching rate and female spawning rate of B. odoriphaga or B. difformis at same heat condition were regarded as the Test variables, while the species were regarded as the Group variables in Independent-Samples T Test comparison.
Similarly, when analysing the enzyme activities and gene expression levels of two Bradysia adults, the MDA concentration, enzyme activities (CAT, SOD, POD and GSTs) and gene expression levels (hsp70 and hsp90) of B. odoriphaga or B. difformis at each heat treatment were regarded as the dependent variable, while the treatments were regarded as the independent variables in one-way ANOVA followed by Tukey's HSD multiple comparisons. With regards to the difference analysis at the same heat conditions, the values of two Bradyisa species were regarded as the dependent variable, while the species (single-sex) were regarded as independent variables in the above-mentioned method. All analyses were performed with PASW Statistics 18.0.0 (2009; SPSS Inc. Quarry Bay, HK). Figures were constructed using SigmaPlot 12.0. Data availability. All data generated or analysed during this study are included in this published article.  Table 3. Primers used in reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR).