Figures
Abstract
The soybean pod borer (Leguminivora glycinivorella Matsumura) successfully survives the winter because of its high expression of 70-kDa heat shock proteins (HSP70s) during its overwintering diapause. The amount of HSP70s is different under different environmental stresses. In this study, inducible heat shock protein 70 and its constitutive heat shock cognate 70 were cloned by RT-PCR and RACE. These genes were named Lg-hsp70 and Lg-hsc70, respectively. Gene transcription and protein expression after cold stress treatment (5°C to −5°C) were analyzed by western blotting and by qRT-PCR for four populations that were sampled in the northeast region of China, including Shenyang, Gongzhuling, Harbin and Heihe, when the soybean pod borer was in diapause. As the cold shock temperature decreased, the levels of Lg-HSP70s were significantly up-regulated. The amount of cold-induced Lg-HSP70s was highest in the southernmost population (Shenyang, 41°50′N) and lowest in the northernmost population (Heihe, 50°22′N). These results support the hypothesis that the soybean pod borer in the northeast region of China displays phenotypic plasticity, and the accumulation of Lg-HSP70s is a strategy for overcoming environmental stress. These results also suggest that the induction of HSP70 synthesis, which is a complex physiological adaptation, can evolve quickly and inherit stability.
Citation: Wang L, Yang S, Han L, Fan D, Zhao K (2014) Phenotypic Plasticity of HSP70s Gene Expression during Diapause: Signs of Evolutionary Responses to Cold Stress among Soybean Pod Borer Populations (Leguminivora glycinivorella) in Northeast of China. PLoS ONE 9(10): e109465. https://doi.org/10.1371/journal.pone.0109465
Editor: Sue Cotterill, St. Georges University of London, United Kingdom
Received: May 2, 2014; Accepted: September 5, 2014; Published: October 15, 2014
Copyright: © 2014 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All Figure 4 files are available from the Genbank database (accession number(s): ACV32641.1 (Helicoverpa zea), BAF03556.1 (Mamestra brassicae), NP_001036892.1 (Bombyx mori), AGQ50301.1 (Leguminivura glycinivurella), XP_966611.1 (Tribolium castaneum), ABP97091.1 (Copris tripartitus), NP_001166228.1 (Nasonia vitripennis), ACD84945.1 (Macrocentrus cingulum), XP_002053707.1 (Drosophila virilis), AAC23392.1 (Ceratitis capitata), AAA37869.1 (Mus musculus), AAK17898.1 (Homo sapiens), XP_003820013.1 (Pan paniscus), AAA17441.1 (Rattus norvegicus), BAK62530.1 (Pan troglodytes), AAD21816.1 (Homo sapiens), CAA70153.1 (Ceratitis capitata), AAR17099.1 (Drosophila virilis), XP_001604951.2 (Nasonia vitripennis), ACD84944.1 (Macrocentrus cingulum), AFE88579.1 (Tenebrio molitor), ABQ39970.1 (Anatolica polita), KF731995 (Leguminivura glycinivurella), BAF69068.1 (Bombyx mori), BAF03555.1 (Mamestra brassicae), ACV32640.1 (Helicoverpa zea)).
Funding: This work is supported by the Special Fund for Modern Agro-industry Technology Research System (CARS-04) and the Fund of Common Wealth Industry (agriculture) Special Research (201103002). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Diapause is a strategy by which insects can escape death and gradually adapt to the environment in response to adverse environmental conditions [1]. Studying the molecular mechanisms of diapause is of great significance because of the important role of diapause in insect survival. Many studies have shown that environmental stress is harmful to insects, even those exposed to sub-zero temperatures over a short period, and that a strong relation between insect death and protein denaturation exists [2]. The expression of HSP70s (70-kDa Heat Shock Proteins) is a normal strategy used by insects to avoid damage arising from environmental stress [3], [4].
As molecular chaperones, HSP70s can recover the normal structure of denatured proteins to repair and protect the cell as well as increase the tolerance and resistance of insects [4], [5], [6]. HSP70s have a conserved structure and prevail in living organisms [7]. Depending on the expression style, these proteins are classified as inducible HSP70 (70-kDa Heat shock protein,HSP70) or constitutive HSC70 (Heat shock cognate protein, HSC70) [8], [9]. In general, the levels of HSP70 are low in normal conditions and increase upon environmental stress, such as high or low temperatures and exposure to heavy metals or microbial infection. HSC70 is stably expressed and only slightly increases upon environmental stress [10].
The expression level of HSP70s can be up-regulated by low temperature during insect diapause. This up-regulation has been observed in Chilo suppressalis and Sesamia nonagrioides, which belong to Lepidoptera [11], [12], in Drosophila triauraria and Drosophila melanogaster, which belong to Diptera [13], [14], and in Leptinotarsa decemlineata, which belongs to Coleoptera [15]. RNAi was used to study the effect of HSP70 expression on cold-stress resistance. Similar to conditions when HSP70 expression was inhibited, Pyrrhocoris apterus has a less intense response to cold resistance in diapause, and the pupae of Sarcophaga crassipalpis have feeble cold resistance [4], [16]. Therefore, we hypothesize that high HSP70 expression plays an important role in increasing insect resistance to cold stress.
HSP70s are an example of adaptive phenotypic plasticity providing inducible tolerance to the environment [17], [18], [19]. Phenotypic plasticity allows insects to react to different environments [20], [21]. This characteristic prevails in different organisms and represents an important aspect of adaptive evolution [22], [23], [24]. Phenotypic plasticity does not only increase cold stress resistance but also allows it to be stably inherited, playing an important role in insect evolution [25], [26], [27]. Because hsp70 is a plasticity gene, understanding the function of hsp70s could supply a good theoretical foundation for understanding insect adaptability to the environment [28], [29], [30]. To date, the homologous genes of hsp70s in relation to phenotypic plasticity have been studied in Rhagoletis cerasi, Calliphora vicina, Lymantria dispar and Melitaea cinxia [19], [31], [32], [33]. However, the variation between conspecifics of different geographical populations has not been widely assessed [34].
The soybean pod borer (Leguminivora glycinivorella Matsumura) belongs to Lepidoptera: Olethreutidae and is one of the most important soybean pests in the world. Only one generation of L. glycinivorella lives in the northeast region of China, which is the main soybean production region, and its diapause stage lasts from October to July [35], [36]. L. glycinivorella can cause severe losses in soybean yield and quality; the loss rate is from 10% to 30% in a general year and up to 50% for more severe situations in a typical year [37], [38]. To date, research concerning L. glycinivorella has primarily focused on environmental ecology and physiology and has not focused on the genetic regulation of the diapause stage. Because a significant difference in climatic conditions exists in the vast, northeast region of China, we presumed that phenotypic plasticity occurred in different populations of L. glycinivorella in response to cold stress. In this study, we cloned Lg-hsp70s and analyzed their expression levels under cold stress induction (5°C to −5°C) to establish a foundation for understanding the molecular regulation of cold resistance and of the ecological adaptability of different populations of L. glycinivorella.
Materials and Methods
Materials
Insect collection sites: The soybean pod borer originated from four different populations: Harbin (Heilongjiang Province, 45°45′N, 126°41′E), Heihe (Heilongjiang Province, 50°22′N, 127°53′E), Gongzhuling (Jilin Province, 43°11′N, 124°02′E) and Shenyang (Liaoning Province, 41°50′N, 123°24′E). The total distance between the south and north populations is approximately 1182 km, and the linear distance from the north to south was approximately 700 km (Figure 1). For our study, we assumed that there was geographical isolation between the different collection sites. The average monthly temperature and the temperature range during diapause are shown in Table 1.
Insect feeding: The adult insects were harvested in soybean fields in late July and reared in a glass on fresh soybean pods, with 20 adults in one glass. The female:male ratio was 1∶1. The glass was placed in a RXZ climate chamber (Ningbo, Jiangsu, China) at 25°C, with a light:dark cycle of 14 h∶10 h. The adults were removed after 12 h, and the eggs were harvested and transferred into a new glass with a soybean pod. After hatching, the larvae were reared to the fourth instar stage in the soybean pod and then harvested in the soybean pod. To induce diapause, the samples were placed in a sterilized soil box in a climate chamber with a mean temperature of 17°C. The temperature was 20°C during the 14-h photophase and 13°C during the 6 h of darkness. The lights were dimmed gradually over 2 h before and after the photophase, imitating sunrise and sunset. After the larvae became cocoons, we transferred the cocoons into a 5°C chamber, where the cocoons overwintered [39]. To diminish the impact of maternal effects, the fourth instar larvae used in the experiment were the second laboratory generation that was collected during the second consecutive year. To test the levels of the Lg-HSP70s, one set consisted of individuals that hatched from eggs collected during the same year that the experiment was performed.
Methods
Cold shock treatment.
The experiment began when the fourth instar larvae of the Heihe population were in the diapause midpoint (150 days). For the cold shock treatment, all fourth instar larvae were exposed to 5°C to −5°C for 1.5 h and then recovered at room temperature (25°C) for 1.5 h (none of the individuals died). Then, the larvae were frozen in liquid nitrogen and stored at −80°C. To determine the constitutive level of HSP70s, the control was a fourth instar larvae maintained at 25°C.
Cloning of Lg-hsp70 and of Lg-hsc70.
Total RNA isolation and first cDNA strand synthesis: RNA was extracted using an RNA isolation kit (Omega, USA), and the concentration and purity were determined using a NanoDrop spectrophotometer (NanoDrop 8000, Parallels, USA) and agarose gel electrophoresis. Then, first-strand cDNA synthesis was performed using a reverse transcription kit (ToYoBo, Japan).
Primer design and gene cloning: Based on the sequences of hsp70 and hsc70 from other insects in the GenBank database, degenerate primers for the conserved region of the gene family were designed using Primer Premier 5.0 software. The primer sequences are listed in Table 2. The PCR reaction for the conserved region was as follows: 3 min at 94°C; 35 cycles of 30 s at 94°C, 30 s at 57.8°C for Lg-hsp70 or 30 s at 58.0°C for Lg-hsc70, 1 min at 72°C; and 10 min at 72°C. Then, the amplified fragment was isolated on a 1.0% agarose gel and ligated into the pMD18-T vector for sequencing. Based on the sequenced fragments of Lg-hsp70 and Lg-hsc70, 5′ RACE and 3′ RACE primers were designed (see Table 2) and used following the manufacturer's protocol for the RACE kits. Full-length Lg-hsp70 and Lg-hsc70 were amplified using the same procedure and used for the following experiment.
Bioinformatics software: The full-length sequences of Lg-hsp70 and Lg-hsc70 were analyzed using DNAStar software. The identity analysis was performed using the BLAST search program, which is available on the NCBI website (http://www.ncbi.nlm.gov/BLAST/). Multiple sequence alignment and phylogenetic analysis were performed using the ClustalW program (http://www.ebiac.uk/clustalw/) and the MEGA 4.1 software with the neighbor-joining method. The PSORT II program (http://psort.hgc.jp/form2.html) was used to calculate the nuclear localization signal fragment. The analyses of protein domains were performed using ProtParam (http://www.cn.expasy.org/tolls/ProtParam), ScanProsite (http://prosite.expasy.org/scanprosite/) and Swiss-Model (http://www.swissmodel.expasy.org/SWISS-MODEL.html) software tools.
Determining the total protein concentration.
The fourth instar larvae were weighed after cold shock. Then, the total protein was isolated using a CytoBuster kit (Novagen, Germany), and the concentration of the protein was determined using a BCA kit (Thermo, USA). The isolated protein was diluted to 2.5 ng/µl in PBS and stored at −80°C. Then, 25 ng of each sample was separated by 12% SDS-PAGE (Mini-PROTEAN Tetra Cell, Bio-Rad). Each treatment was performed in 3 replicates, and β-actin (approximately 42 kDa) was used as a reference.
After SDS-PAGE, the sample was electroblotted onto a PVDF membrane and blocked in 5% dried skim milk.The membrane was incubated with a mouse monoclonal HSP70 antibody that could identify both HSP70 and HSC70 (H5147, Sigma, USA) (1∶5000, V:V) and with a mouse monoclonal β-actin antibody (1∶5000, V:V) (A5441, Sigma, USA) at 4°C overnight. After the membrane was washed three times in TBST for 10 min each, goat anti-mouse lgG (H+L) was used as the secondary antibody (Sigma, USA) (1∶1000, V:V) at room temperature for 2 h and then washed three times in TBST for 10 min each. The blots were developed in DAB and photographed. The bandwidth and gray level were analyzed using ImageJ 4.0 software. Then, a one-way ANOVA was computed using SPSS 16.0 software, with P<0.05.
Quantitative real-time PCR.
qRT-PCR primers were designed using the Lg-hsp70 and Lg-hsc70 sequences (see Table 2). The products were 179 bp and 141 bp, respectively. Using β-actin as the reference gene, the primers were designed according to Li, and the amplified fragment was 132 bp [40]. Total RNA was extracted using an RNA isolation kit (Omega, USA), and the concentrations were brought to 100 ng/µl using DEPC-treated water in all samples. Then, cDNA synthesis was performed using a reverse transcription kit (ToYoBo, Japan).
Standard curve: Using the first-strand cDNA as a template and the above-mentioned primers for PCR amplification, the PCR products were purified using a Gel Extraction Kit (Omega, USA), ligated into the pMD18-T vector (TaKaRa, Japan) and transformed into Escherichia coli competent cells with blue-white color selection. Positive clones were harvested and cultured overnight. The plasmid was extracted and identified by PCR. Then, the positive clones were sequenced at Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). The correctly identified plasmid was quantified using a NanoDrop (NanoDrop 8000, Parallels, USA) and used at a 10x dilution as the standard for the qRT-PCR reaction. Each sample had 3 replicates. The standard equation was derived based on the critical number of cycles (threshold cycle, Ct) and on the logarithmic values of the initial template copy number. The qRT-PCR reaction used a fluorescent dye, SYBR Green I. The qRT-PCR reaction conditions were as follows: 95°C denaturation 30 s, 95°C denaturation 10 s, and 40 cycles of 60°C annealing and extension for 30 s. The Ct value of the reaction was read by the Opticon 3 software.
Data processing: The data were calculated using the 2-ΔΔCt method after standard genetic homogenization treatment. For the control, the value of ΔΔCt was 0 (20 = 1). Statistical significance was determined using a one-way ANOVA and post-hoc Duncan multiple range tests with the SPSS 16.0 software. The significance was established at P<0.05.
Results
Sequence analysis of Lg-hsp70 and Lg-hsc70
Lg-hsp70 and Lg-hsc70 were cloned and deposited into the GenBank database under accession numbers KF731995 and KC844150, respectively. The coding amino acids are shown in Figures 2 and 3. The full-length Lg-hsp70 sequence was 2253 bp [excluding poly(A)] and included a 102-bp 5′-UTR, a 189-bp 3′-UTR, and a 1941-bp ORF, which encoded 646 aa. The molecular weight was 70.73 kDa, and the PI was 5.54. The positive charge of disability (Arg + Lys) was 83, and the negative charge of disability (Asp + Glu) was 93. In addition, the full-length Lg-hsc70 sequence was 2147 bp [excluding poly(A)] and included a 102-bp 5′-UTR, a 59-bp 3′-UTR and a 1986-bp ORF, which encoded 661 aa. The molecular weight was 73.23 kDa, and the PI was 5.30. The positive charge of disability (Arg + Lys) was 76, and the negative charge of disability (Asp + Glu) was 101. The protein sequences showed that both Lg-HSP70 and Lg-HSC70 have 3 conserved HSP70 family domains, EEVD at the end of the C-terminus [41] and two putative bipartite nuclear localization signal (NLS) sequences (Figures 1 & 2).
The upper sequences indicate the nucleotides, and the lower sequences show the inferred amino acids. Start and stop codons are in bold. The signature sequences of the HSP70s family are shown in boxes, the nuclear localization signal sequence is underlined, the consensus sequence EEVD at the C-terminus is indicated in italics and the consensus polyadenylation signal (AATAAA) is shown in a gray box.
The upper sequences indicate the nucleotides, and the lower sequences show the inferred amino acids. The start and stop codons are in bold. The signature sequences of the HSP70s family are shown in boxes, the nuclear localization signal sequence is underlined, the consensus sequence EEVD at the C-terminus is indicated in italics and the consensus polyadenylation signal (AATAAA) is shown in a gray box.
Phylogenetic analysis of Lg-HSP70 and Lg-HSC70
Using homology analysis, we found that Lg-HSP70 was more than 73.6% similarity with other known HSP70; in particular, it had 89.5% identity with Bombyx mori (BAF69068) and Mamestra brassicae (BAF03555) and 70.6% identity with vertebrates. Lg-HSC70 had more than 78.1% similarity with other known HSC70s, and it had 86% identity with Helicoverpa zea (ACV32641) and 72.6% identity with vertebrates. A phylogenetic tree was constructed, revealing that the HSP70 family could be divided into two main branches (Figure 4). One branch is HSC70, which also contains the other 11 HSC70 genes, and the other branch is HSP70, which contains all other HSP70 genes. Therefore, we could confirm that Lg-hsc70 and Lg-hsp70 are constitutive and inducible genes, respectively.
A three-letter code has been included to indicate the order name of the corresponding insects and vertebrates (COL = Coleoptera, LEP = Lepidoptera, DIP = Diptera, HYM = Hymenoptera and VER = Vertebra). The values indicated on the branches correspond to bootstrap percentages (BP).
Expression of the Lg-HSP70 genes
The transcription and expression of Lg-HSP70s were determined using qRT-PCR and western blotting, respectively. The results showed that the levels of the Lg-HSP70s were not significantly different (P>0.05) between field- and laboratory-derived populations in the four cities. Three factors were considered to analyze the relationships among temperature (cold stress and control), sex (female and male) and population (50°22′N, 45°45′N, 43°11′N and 41°50′N). However, only the treatment between temperature and population had a significant effect on the Lg-HSP70 levels (P<0.05). Western blotting showed that the expression of Lg-HSP70s that originated from the same geographical population was significantly up-regulated as the temperature decreased, and it peaked at -5°C (Figure 5). Compared with the amount of Lg-HSP70s at the same temperature, there was a significant increasing trend as the latitude decreased. Shenyang (41°50′N) expressed the most Lg-HSP70s, and Heihe (50°22′N) expressed the least Lg-HSP70s. Because no differences in the HSP70 level were observed, we concluded that the laboratory-rearing conditions had little effect on the HSP70 level. These results revealed that cold stress has a significant inducible effect on the expression of Lg-HSP70s, indicating that phenotypic plasticity variation for Lg-HSP70s occurs in different geographical populations.
(A) Western blotting of Lg-HSP70s. 1: Shenyang, 2: Gongzhuling, 3: Harbin, 4: Heihe. (B) Relative gray levels of Lg-HSP70s (*: significant difference compared with control, P<0.05).
qRT-PCR analysis: The positive clones were identified by PCR. The soybean pod borer cDNA sequences for Lg-hsp70, Lg-hsc70 and β-actin had 100% homology, indicating that the recombinant plasmid contained the target fragment. Using a UV spectrophotometer, the standard concentrations of Lg-hsp70, Lg-hsc70 and β-actin were found to be 22 ng/µl, 18 ng/µl and 15 ng/µl, respectively. The copy numbers of the Lg-hsp70, Lg-hsc70 and β-actin standards were 7.01×109, 5.79×109 and 4.87×109 copies/µl, respectively. The equations of the standard curves of Lg-hsp70, Lg-hsc70 and β-actin were Y = -3.124X+40.601, Y = -3.124X+46.327 and Y = -3.297X+39.876, respectively, where Y represents the value of Ct, and X represents the logarithmic value of the template. The correlation coefficients were R2 = 0.992, R2 = 0.998 and R2 = 0.997, respectively. When the template cDNA was diluted, the Ct value was up-regulated 3 to 4 cycles, and the concentration decreased with each 10-fold dilution. The slopes of three standard curves were near the expected value of -3.322, with a correlation coefficient close to 1.000, suggesting that the results of the qRT-PCR could be used for the quantitative detection of Lg-hsp70 and Lg-hsc70 over a wide range.
Expression of Lg-hsp70 induced by cold shock: Compared with the control (25°C), the expression level of Lg-hsp70 could be induced in the four populations at 5°C. Lg-hsp70 increased 7.08 fold in Shenyang, which was significantly higher than the levels in the other populations, and there was no significant difference among the other three populations. At 0°C, the Lg-hsp70 transcripts were present at a high level in each population, and there was a significant difference in their levels compared with the level of the control (P<0.05). The expression of Lg-hsp70 in each population reached its peak at –5°C, increasing by 80.18 fold, 69.32 fold, 41.37 fold and 25.01 fold in Shenyang, Gongzhuling, Harbin and Heihe, respectively, compared with the control. A significant difference existed among the different geographical populations (P<0.05) (see Table 3). Thus, cold treatment had a significant effect on inducing Lg-hsp70 at diapause.
Expression of Lg-hsc70 induced by cold shock: There was a small, inducible effect on the transcription of Lg-hsc70 by cold treatment from −5°C to 5°C, and the level was significantly lower than that of Lg-hsp70. At 5°C, the expression level of Lg-hsc70 did not differ from the control. At 0°C, the expression level of Lg-hsc70 significantly increased (P<0.05); however, there was no significant difference between Gongzhuling and Harbin (P>0.05). The highest transcription of Lg-hsc70 was observed at −5°C, increasing by 7.91 fold, 5.09 fold, 4.67 fold and 2.07 fold in Shenyang, Gongzhuling, Harbin and Heihe, respectively, compared with the control. A significant difference existed among the four geographical populations (see Table 3).
Discussion
The Lg-HSP70 and Lg-HSC70 sequences both contain a conserved amino acid site, and the consensus sequence EEVD at the end of the C-terminus revealed that the two proteins belong to the DanK cytoplasmic-type protein of the HSP70 family. In addition, two nuclear localization sites (NLS) that play important roles in the inhibitory effect of HSP70s against nucleolar impairment induced by oxidative stress were identified [42].
To determine whether Lg-hsp70 and Lg-hsc70 were inducible or constitutive, a cluster analysis was performed. The results showed that Lg-hsp70 and Lg-hsc70 belong to two different branches of hsp70 and hsc70, respectively. Some recent studies have reported that the expression level of hsp70 increases significantly after induction by environmental stress but that hsc70 is expressed with a time lag and with a low range compared with hsp70 [43]. In our experiment, the expression time and rate of increase of Lg-hsp70 significantly differed from that of Lg-hsc70, which supports the conclusion that Lg-hsp70 is an inducible gene and that Lg-hsc70 is a constitutive gene. The difference in expression of the two genes may be because the Lg-hsp70 gene has no intron and Lg-hsc70 does, which affects the split joint of the mRNA [44], [45].
Selection for low-temperature stress: Although L. glycinivorella overwinters in buried soil, this insect can still be exposed to harmful environmental temperatures. In northeastern China, the depth of the frozen soil varies with year and location [46]. Consequently, the optimum overwinter depth for L. glycinivorella is unpredictable, and exposure to the cold is possible. According to the temperature data for frozen soil from the northeastern region and the report that −5°C was the lethal temperature for L. glycinivorella to overwinter [47], −5°C and −10°C were designed as the pretreatment temperatures in this study. The results showed that all larvae were dead at −10°C; therefore, −5°C was selected as the treatment temperature.
Lg-HSP70 protein expression and mRNA transcription were analyzed after cold stress in the four geographical populations. The results revealed that HSP70s had a strong relation with insect resistance to cold stress [11], [34]. Thus, this mechanism is most likely a strategy that L. glycinivorella uses to react to cold stress. Additionally, we found that the up-regulation of Lg-HSP70s varies with latitude. A southern population may have less chance of experiencing cold stress during their overwintering diapause compared with a northern population [48]. Some studies have shown that the production of HSP70s as a response to temperature shock is less intense in organisms that are more frequently exposed to unfavorable temperatures in their habitat compared with those organisms exposed to benign conditions [49]. Research concerning Leptinotarsa decemlineata showed the same trend when comparing a southern population (Bonin, Poland, 54°N) to a northern population (Lodeynoye Pole, Russia, 60°N) [50].
Because HSP70 production requires more energy, this production may incur significant costs to the synthesis of other proteins [51], [52]. Therefore, the northern population might have evolved other more reasonable, less costly mechanisms to cope with cold stress [48], [53]. In recent years, possible mechanisms have been studied and include accumulating low-molecular-weight cryoprotectants [54], synthesizing antifreeze proteins [55] and remodeling the structure of the cell membrane [56]. In contrast, with the development of molecular biology, the function, reactions and location of HSP70s also could be identified by bioinformatics tools and proteomics techniques, such as 2-dimensional electrophoresis and mass spectra which have become powerful tools to discover and validate protein-coding genes. Some proteomic studies showed that different isoforms of the HSP70 family, including HSP68, HSP70Ba, and HSP70Bb, exist and that several of them displayed strong increases in protein abundance during environmental variations [57], [58]. Furthermore, the gene annotation and localization might play a pivotal role to facilitate identification and analysis of HSP70s in the integrated genome of L. glycinivorella. Thus, on the basis of this research, application of proteomic and genomic strategies could supply good support for the relevant mechanism of L. glycinivorella from different populations.
L. glycinivorella from different populations showed a differential response to cold stress. The significant differences in the expression levels of LgHSP70s in the southern (Shenyang, 41°50′N) and northern populations (Heihe, 50°22′N) showed strong evidence for phenotypic plasticity among the four different populations [59]. The phenotypic plasticity in these populations contributes to the soybean pod borer response to different selective pressures under different environmental conditions and has great potential for improving their ability to resist environmental stress [60], [61]. Because relative reports of the damage of L. glycinivorella date to before the 1950s [38], we assume that there must be a long history of L. glycinivorella damaging the soybean crop in northeastern China. Although no reports of genetic variability can be found, we believe that phenotypic plasticity variation based on epigenetic mechanisms has occurred for a long period in different geographic populations of L. glycinivorella so that they could adapt to the different ecological conditions. Thus, because this variation can be stably inherited, it will undoubtedly have a profound effect on population adaptability [62], [63], [64].
Acknowledgments
We gratefully acknowledge Dr. Xin Dawei (Northeast Agriculture University, China) for his constructive reviews of the manuscript.
Author Contributions
Conceived and designed the experiments: LW SY LH DF KZ. Performed the experiments: LW SY. Analyzed the data: LW SY. Contributed reagents/materials/analysis tools: LW SY LH DF KZ. Wrote the paper: LW SY KZ.
References
- 1. Danks HV (2007) The elements of seasonal adaptations in insects. The Canada Entomologist 139(1): 1–44.
- 2.
Watson P, Morris G (1987) Cold shock injury in animals. In: Bowler K, Fuller B, eds. Temperature and animal cells. Cambridge: The Company of Biologist Limited. pp 311–340.
- 3. Bettencourt BR, Hogan CC, Nimali M, Drohan BW (2008) Inducible and constitutive heat shock gene expression responds to modification of Hsp70 copy number in Drosophila melanogaster but does not compensate for loss of thermotolerance in Hsp70 null flies. BMC Biology 6(5): 1–15.
- 4. Rinehart JP, Li A, Yocum GD, Robich RM, Hayward SA, et al. (2007) Up-regulation of heat shock proteins is essential for cold survival during insect diapause. Proceedings of the National Academy of Sciences of the United States of America 104: 11130–11137.
- 5. Mads D (2007) The heat shock protein 70 family: Highly homologous proteins with overlapping and distinct functions. FEBS Letters 581: 3702–3710.
- 6. Doucet D, Walker VK, Qin W (2009) The bugs that came in form the cold: molecular adaptations to low temperatures in insects. Cellular and Molecular life Sciences 66(8): 1404–1418.
- 7. Schlesinger MJ (1990) Heat shock proteins. The Journal of Biological Chemistry 265: 12111–12114.
- 8. Qin W, Tyshenko MG, Wu BS, Walker VK, Robertson RM (2003) Cloning and characterization of a member of the Hsp70 gene family from Locusta migratoria, a highly thermotolerant insect. Cell Stress and Chaperones 8: 144–152.
- 9. Franzellitti S, Fabbri E (2005) Differential HSP70 gene expression in the Mediterranean mussel exposed to various stressors. Biochemical and Biophysical Research Communications 336(4): 1157–163.
- 10. Daugaard M, Rohde M, Jäättela M (2007) The heat shock protein 70 family: highly homologous proteins with overlapping and diatinct functions. FEBS Letters 581(19): 3702–3710.
- 11. Sonoda S, Fukumoto K, Izumi Y, Yoshida H, Tsumuki H (2006) Cloning of heat shock protein genes (hsp90 and hsc70) and their expression during larval diapause and cold tolerance acquisition in the rice stem borer, Chilo suppressalis Walker. Archives of Insect Biochemistry and Physiology 63(1): 36–47.
- 12. Gkouvitsas T, Kontogiannatos D, Kourti A (2009) Cognate Hsp70 gene is induced during deep larval diapause in the moth Sesamia nonagrioides. Insect Molecular Biology 18(2): 253–264.
- 13. Goto SG, Yoshida KM, Kimura MT (1998) Accumulation of Hsp70 mRNA under environmental stresses in diapausing and nondiapausing adults of Drosophila triauraria. Journal of Insect Physiology 44: 1009–1015.
- 14. Sinclair BJ, Gibbs AG, Roberts SP (2007) Gene transcription during exposure to, and recovery from, cold and desiccation stress in Drosophila melanogaster. Insect Molecular Biology 16: 435–443.
- 15. Yocum GD (2001) Differential expression of two HSP70 transcripts in response to cold shock, thermoperiod, and adult diapause in the Colorado potato beetle. Journal of Insect Physiology 47: 1139–1145.
- 16. Kostál V, Tollarová-Borovanská M (2009) The 70 kDa heat shock protein assists during the repair of chilling injury in the insect, Pyrrhocoris apterus. PLoS One 4(2): e4546.
- 17. Callahan HS, Maughan H, Steiner UK (2008) Phenotypic Plasticity, Costs of Phenotypes, and Costs of Plasticity. Annals of the New York Academy of Sciences 1133: 44–46.
- 18. Schulte PM, Healy TM, Fangue NA (2011) Thermal performance curves, phenotypic plasticity, and the time scales of temperature exposure. Integrative and Comparative Biology 51(5): 691–702.
- 19. Luo SQ, Wong SC, Xu CR, Hanskic I, Wang RJ, et al. (2014) Phenotypic plasticity in thermal tolerance in the Glanville fritillary butterfly. Journal of Thermal Biology 42: 33–39.
- 20. Bradshaw AD (1965) Evolutionary significance of phenotypic plasticity in plants. Advances in Genetics 13: 115–155.
- 21. Van Kleunen M, Fischer M (2005) Constrains on the evolution of adaptive phenotypic plasticity in plants. New Phytologist 166: 49–60.
- 22. Sultan SE (2004) Promising directions in plant phenotypic plasticity. Perspectives in Plant Ecology 6(4): 227–233.
- 23. Pigliucci M, Murren CJ, Schlichting CD (2006) Phenotypic plasticity and evolution by genetic assimilation. Journal of Experimental Biology 209: 2362–2367.
- 24. Bourgeois M, Jacquin F, Savois V, Sommerer N, Labas V, et al. (2009) Dissecting the proteome of pea mature seeds reveals the phenotypic plasticity of seed protein composition. Proteomics 9(2): 254–271.
- 25. Whitman DW, Ananthakrishnan TN (2009) Phenotypic plasticity of insects: mechanisms and consequences. pp. x+ 894 pp
- 26. Stillwell RC, Blanckenhorn WU, Teder T, Davidowitz G, Fox CW (2010) Sex differences in phenotypic plasticity affect variation in sexual size dimorphism in insects: from physiology to evolution. Annual Review of Entomology 55: 227–245.
- 27. Schlichting CD, Wund MA (2014) Phenotypic plasticity and epigenetic marking: an assessment of evidence for genetic accommodation. Evolution 68(3): 656–672.
- 28. Zhou XZ, Tarver MR, Scharf ME (2007) Hexamerin-based regulation of juvenile hormone-dependent gene expression underlies phenotypic plasticity in a social insect. Development 134: 601–610.
- 29. Moczek AP (2010) Phenotypic plasticity and diversity in insects. Philosophical Transactions of the Royal Society B: Biological Sciences 365(1540): 593–603.
- 30. Morris M, Rogers SM (2014) Integrating phenotypic plasticity within an ecological genomics framework: recent insights from the genomics, evolution, ecology, and fitness of plasticity. Advances in Experimental Medicine and Biology Volume 781: 73–105.
- 31. Moraiti CA, Nakas CT, Papadopoulos NT (2014) Diapause termination of Rhagoletis cerasi pupae is regulated by local adaptation and phenotypic plasticity: escape in time through bet-hedging strategies. Journal of Evolutionary Biology 27(1): 43–54.
- 32.
Coleman PC, Bale JS, Hayward SA (2014) Cross generation plasticity in cold hardiness is associated with diapause, but not the non-diapause developmental pathway, in the blowfly, Calliphora vicina. The Journal of Experimental Biology http://dx.doi: 10.1242/jeb.098053 .
- 33. Mrdaković M, Stojković B, Perić-Mataruga V, Ilijin L, Vlahović M, et al. (2014) Adaptive phenotypic plasticity of gypsy moth digestive enzymes. Central European Journal of Biology 9(3): 309–319.
- 34. Zhang Q, Denlinger DL (2010) Molecular characterization of heat shock protein 90, 70 and 70 cognate cDNAs and their expression patterns during thermal stress and pupal diapause in the corn earworm. Journal of Insect Physiology 56(2): 138–150.
- 35. Zhao GY, Wang J, Han YP, Teng WL, Sun GL, et al. (2008) Identification of QTL underlying the resistance of soybean to pod borer, Leguminivora glycinivorella (Mats.) obraztsov, and correlations with plant, pod and seed traits. Euphytica 164 (1): 275–282.
- 36. Zhang T, Feng J, Cai C, Zhang X (2011) Synthesis and field test of three candidates for soybean pod borer's sex pheromone. Natural Product Communications 6(9): 1323–1326.
- 37. Hu DH, He J, Zhou YW, Feng JT, Zhang X (2012) Synthesis and Field Evaluation of the Sex Pheromone Analogues to Soybean Pod Borer Leguminivora glycinivorella. Molecules 17(10): 12140–12150.
- 38. Wang KQ, Mei XR, Liu XL, Shao TY, Li XM, et al. (2013) Field trapping effect of two types of sex pheromone lures and traps of Leguminivora glycinivorella. Heilongjiang Agricultural Sciences 5: 34–36 (in Chinese).
- 39. Zang LS, Song LV, Yuan XH, Ruan CQ, Shi SS (2012) Parasitism activity of four indigenous Trichogramma species on eggs of different ages of the soybean pod borer, Leguminivora glycinivorella. Acta Phytophylacica Sinica 39(4): 381–382 (in Chinese).
- 40. Li DM, Meng FL, Wang ZK, Zang ZY, Sui J, et al. (2012) Cloning and characterization of 28SrDNA gene form soybean pod borer (Leguminivora glycinivorella Matsumurav). Journal of Northeast Agricultural University 43(7): 13–17 (in Chinese).
- 41. Demand J, Lüders J, Höhfeld J (1998) The carboxy-terminal domain of Hsc70 provides binding sites for a distinct set of chaperone cofactors. Molecular Cell Biology 18(4): 2023–2028.
- 42. Wang KK, E SM, Jiang L, Zhang HL, Liu K, et al. (2005) Roles of nuclear localization signal (NLS) in inhibitory effect of HSP70 on nucleolar segregation induced by oxidative stress. Progress Biochemistry Biophysics 32(5): 456–462 (in Chinese).
- 43. Deane EE, Woo NYS (2005) Cloning and characterization of the hsp70 Multigene family from silver sea bream: modulated gene expression between warm and cold temperature acclimation. Biochemical and Biophysical Research Communications 330(3): 776–783.
- 44. Yost HJ, Lindquist S (1991) Heat shock proteins affect RNA processing during the heat shock response of Saccharomyces cerevisiae. Molecular and Cellular Biology 11: 1062–1068.
- 45. Chuang K, Ho S, Song Y (2007) Cloning and expression analysis of heat shock cognate 70 gene promoter in tiger shrimp (Penaeus monodon). Gene 405: 10–18.
- 46. Zhang W, Song SY, Jing QM, Li H, Sha HL (2005) Identification of resistance of new soybean varieties (Lines) to soybean pod borer in Jilin Province. Journal of Jilin Agricultural Sciences 30(6): 40–41 (in Chinese).
- 47. Xu QF, Guo SG, Han YM, Feng Z, Zhang R, et al. (1965) A study on the soybean pod borer (Leguminivora glycinivorella (Mats.) Obraztsov). Acta Entomologlca sinica 14(5): 461–475 (in Chinese).
- 48. Lansing E, Justesen J, Loeschcke V (2000) Variation in the expression of Hsp70, the major heat-shock protein, and thermotolerance in larval and adult selection lines of Drosophila melanogaster. Journal of Thermal Biology 25: 443–450.
- 49. Sørensen J, Dahlgraad J, Loeschcke V (2001) Genetic variation in thermal tolerance among natural populations of Drosophila buzzatii: down regulation of Hsp70 expression and variation in heat stress resistance traits. Functional Ecology 15: 289–296.
- 50. Lyytinen A, Mappes J, Lindström L (2012) Variation in Hsp70 Levels after Cold Shock: Signs of Evolutionary Responses to Thermal Selection among Leptinotarsa decemlineata Populations. PLoS One 7(2): e31446.
- 51. Krebs RA, Loeschcke V (1994) Costs and benefits of activation of the heat-shock response in Drosophila melanogaster. Functional Ecology 8: 730–737.
- 52. Feder JH, Rossi JM, Solomon J, Solomon N, Lindquist S (1992) The consequences of expressing hsp70 in Drosophila cells at normal temperatures. Genes and Development 6: 1402–1413.
- 53. Sørensen J, Kristensen TN, Loeschcke V (2003) The evolutionary and ecological role of heat shock proteins. Ecology Letters 6: 1025–1037.
- 54. Crowe JH, Crowe LM, Carpenter JF, Rudolph AS, Wistrom CA, et al. (1988) Interactions of sugars with membranes. Biochimicaet Biophysica Acta 947: 367–384.
- 55. Duman JG (2001) Antifreeze and ice nucleator proteins in terrestrial arthropods. Annual Review of Physiology 63: 327–357.
- 56. Tomcala A, Tollarová M, Overgaard J, Simek P, Kostál V (2006) Seasonal acquisition of chill-tolerance and restructuring of membrane glycerophospholipids in an overwintering insect: triggering by low temperature, desiccation and diapause progression. Journal of Experimental Biology 209: 4102–4114.
- 57. Aparna HS, Rajesh RK, Manjunatha HB (2010) Molecular characterization of heat shock proteins 90 (HSP83?) and 70 in tropical strains of Bombyx mori. Proteomics 10: 2734–2745.
- 58. Hervé C, Johannes O, Emmanuelle C, Jesper GS (2013) Proteomic profiling of thermal acclimation in Drosophila melanogaster. Insect Biochemistry and Molecular Biology 43: 352–365.
- 59. Schlichting CD, Smith H (2002) Phenotypic plasticity: linking molecular mechanisms with evolutionary outcomes. Evolutionary Ecology 16: 189–211.
- 60. Ghalambor CK, Mckay JK, Carroll SP, Reznick DN (2007) Adaptive versus non-adaptive phenotypic plasticity and the potential for contemporary adaptation in new environments. Functional Ecology 21: 394–407.
- 61. Sexton JP, McKay JK, Sala A (2002) Plasticity and genetic diversity may allow saltcedar to invade cold climate in North America. Ecological Applications 12: 1652–1660.
- 62. King EG, Roff DK (2010) Modeling the Evolution of Phenotypic Plasticity in Resource Allocation in Wing-Dimorphic Insects. The American Naturalist 175(6): 702–716.
- 63. Moczek AP, Kijimoto T, Snell-Rood E, Rocha G, Pespeni M, et al. (2014) Evolutionary and ecological genomics of developmental plasticity: novel approaches and first insights from the study of horned beetles. Ecological Genomics 781: 127–148.
- 64. Tobin PC, Parry D, Aukema BH (2014) The influence of climate change on insect invasions in temperate forest ecosystems. Forestry Sciences 81: 267–293.