Molecular characterization and expression of six heat shock protein genes in relation to development and temperature in Trichogramma chilonis

Trichogramma is a kind of egg parasitoid wasp that is widely used to control lepidopterous pests. Temperature is one of the main factors that determines the various life activities of this species, including development, reproduction and parasitism efficiency. Heat shock proteins (HSPs) are highly conserved and ubiquitous proteins that are best known for their responsiveness to temperature and other stresses. To explore the potential role of HSPs in Trichogramma species, we obtained the full-length cDNAs of six HSP genes (Tchsp10, Tchsp21.6, Tchsp60, Tchsp70, Tchsc70-3, and Tchsp90) from T. chilonis and analyzed their expression patterns during development and exposure to temperature stress. The deduced amino acid sequences of these HSP genes contained the typical signatures of their corresponding protein family and showed high homology to their counterparts in other species. The expression levels of Tchsp10, Tchsp21.6 and Tchsp60 decreased during development. However, the expression of Tchsc70-3 increased from the pupal stage to the adult stage. Tchsp70 and Tchsp90 exhibited the highest expression levels in the adult stage. The expression of six Tchsps was dramatically upregulated after 1 h of exposure to 32 and 40°C but did not significantly change after 1 h of exposure to 10 and 17°C. This result indicated that heat stress, rather than cold stress, induced the expression of HSP genes. Furthermore, the expression of these genes was time dependent, and the expression of each gene reached its peak after 1 h of heat exposure (40°C). Tchsp10 and Tchsp70 exhibited a low-intensity cold response after 4 and 8 h of exposure to 10°C, respectively, but the other genes did not respond to cold at any time points. These results suggested that HSPs may play different roles in the development of this organism and in its response to temperature stress.

Introduction temporal expression patterns of six Tchsps were also investigated during cold (10˚C) and heat (40˚C) exposure. To our knowledge, this is the first report on the isolation and analysis of hsps from T. chilonis. Our results are expected to help elucidate the potential contribution of these HSPs to thermal tolerance and development.

Insects
Prepupae of T. chilonis (parasitized eggs) and eggs of Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae) were obtained from the Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences, People's Republic of China. T. chilonis cultures were maintained on irradiated C. cephalonica eggs for several generations at 25 ± 1˚C with 75 ± 5% relative humidity and a 14 L:10 D photoperiod.

Sampling at different developmental stages
The irradiated eggs of C. cephalonica were glued on 4 paper cards (2 × 1 cm) and exposed to freshly emerged T. chilonis for 30 min. These egg cards were transferred to different glass cylinders and maintained at 25 ± 1˚C with 75 ± 5% relative humidity and a 14 L:10 D photoperiod. Parasitized eggs on different cards were dissected to collect the larvae, prepupae, pupae and adults of T. chilonis. The developmental stages of T. chilonis were confirmed under a stereoscope as described in previous studies [28,29]. Every 6 h, a small number of parasitized eggs were dissected to determine the developmental stage of T. chilonis. At the larval stage of T. chilonis, the colors of individuals and parasitized eggs were both white. Larvae with oval shapes were collected. At the prepupal stage, the color of parasitized eggs was black, and pulm spots were visible on the body. Prepupae were collected when pulm spots disappeared from the head and tail. At the pupal stage, the color of parasitized eggs turned deep, red compound eyes appeared on the body, and pulm spots disappeared. Pupae with small black spots on their bodies were collected. The adults were collected once they emerged from the eggs. To collect corresponding individuals, parasitized eggs were immediately placed on a filter-paper soaked with Sample Protector for RNA/DNA (TaKaRa, Dalian, China) and dissected under a stereoscope. The specimens were immediately transferred to TRIzol (Invitrogen, Darmstadt, Germany) and stored in a -80˚C refrigerator. Fifty wasps from each developmental stage were collected. The experiment was repeated three times.

Temperature exposure
Considering that T. chilonis wasps are often released as pupae inside the host eggs, these parasitized eggs were chosen for temperature exposure experiments. The parasitized eggs were exposed to temperatures of 10, 17, 32 and 40˚C for 1 h, and parasitized eggs kept at 25˚C were collected as controls. These parasitized eggs were then dissected to collect wasps under a stereoscope. In addition, wasps were collected at different time points (1, 2, 4 and 8 h) during cold (10˚C) and heat (40˚C) exposure. The sampling method was the same as described above.

Cloning the full-length cDNA of hsps
Total RNA from adults was isolated with a TRIzol Reagent Kit according to the supplier's instructions. Assessment of the quality and quantity of total RNA was performed by electrophoresis and with a NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA). Firststrand cDNA was generated with a PrimeScript TM RT Reagent Kit (TaKaRa, Dalian, China). Templates for 5' and 3' RACE were constructed using a SMART TM RACE cDNA Amplification Kit (Clontech, California, USA). Primers (S1 Table) were designed based on the nucleotide sequences from the transcriptome data of T. chilonis (SRA accession number: SRP119024). PCR products were cloned and then sequenced by Sangon (Shanghai).

Bioinformatics analysis
Using the DNASTAR software package, full-length cDNAs of hsps were obtained based on the sequenced fragments. The BLAST search was performed to find homologous sequences in GenBank. Multiple sequence alignment and identity analysis were performed using DNA-MAN software. The open reading frame (ORF) and deduced amino acid sequence of each hsp were identified and obtained using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The predicted molecular weight and theoretical isoelectric point (pI) of the deduced proteins were predicted with the ExPASy (http://www.expasy.org/). Domains were predicted by SMART tool (http://smart.embl-heidelberg.de/). Phylogenetic analysis was performed using MEGA software (version 6.0) with the 1000 bootstrap replicates. Five neighbor-joining (NJ) phylogenetic trees were constructed using members of HSP10, sHSPs, HSP60, HSP70 and HSP90 familiy.

Quantitative real-time PCR
Total RNA of the samples from each treatment was extracted and reverse transcribed as described above. Primers were designed based on the conserved regions of the hsps, and glyceraldehyde-3-phosphate dehydrogenase (gapdh) was used as the control (S1 Table). The realtime PCR reaction was performed in a 10 μL reaction volume following the manufacturer's protocol for SYBR 1 Premix Ex Taq™ (TaKaRa, Dalian, China). The expression profiles of hsps were determined on a Roche 480 Real-Time PCR System (Roche, Switzerland) under the following conditions: 95˚C for 3 min, 40 cycles of 95˚C for 10 s, 60˚C for 20 s and 72˚C for 20 s. The melting curve analysis was applied to ensure the specificity of primers at the end of the program. The relative abundance of each hsp was calculated according to the 2 −ΔΔCt method [30].

Statistics
The expression values of the hsps are presented as the means ± SEM. Statistical analysis was performed by SPSS v. 16.0 software (SPSS, Chicago, IL, USA) with one-way analysis of variance (ANOVA) and Duncan's post hoc tests.

Characterization of hsp genes
Tchsp10.. The full-length cDNA of Tchsp10 was 705 bp, including an ORF of 315 bp, a 5'untranslated region (UTR) of 205 bp and a 3'-UTR of 185 bp (GenBank accession number MH490973). The ORF of Tchsp10 encoded a polypeptide of 104 amino acids with a predicted molecular weight of 11.26 kDa and a pI of 8.93. TcHSP10 showed topical Cpn10 superfamily characteristics with a conserved domain (aa 9-102) and and a mobile loop (aa 25-38) (Fig 1).
Tchsp21.6.. The full-length cDNA of Tchsp21.6 was 2119 bp, including an ORF of 576 bp, a 5'-UTR of 218 bp and a 3'-UTR of 1325 bp (GenBank accession number MH490974). The ORF of Tchsp21.6 encoded a polypeptide of 191 amino acids with a predicted molecular weight of 21.68 kDa and a pI of 5.6. TcHSP21.6 was a typical small HSP, containing a metazoan αcrystalline domain (ACD) (Fig 2).
Tchsp60. The full-length cDNA of Tchsp60 was 2222 bp, including an ORF of 1716 bp, a 5'-UTR of 197 bp and a 3'-UTR of 309 bp (GenBank accession number MH490975). The ORF of Tchsp60 encoded a polypeptide of 571 amino acids with a predicted molecular weight of 60.48 kDa and a pI of 5.18. TcHSP60 contained a classical mitochondrial HSP60 signature motif (AAVEEGIVPGGG), a C-terminal Gly-Gly-Met repeat (GGM repeat motif) and ATP/ ADP binding sites (Fig 3).
Two TcHSP70 genes. The full-length cDNA of Tchsp70 was 2573 bp, including an ORF of 1992 bp, a 5'-UTR of 13 bp and a 3'-UTR of 568 bp (GenBank accession number MH490976). The ORF of Tchsp70 encoded a polypeptide of 663 amino acids with a predicted molecular weight of 73.26 kDa and a pI of 5.62.
The full-length cDNA of Tchsc70-3 was 2668 bp, including an ORF of 1992 bp, a 5'-UTR of 186 bp and a 3'-UTR of 490 bp (GenBank accession number MH490977). The ORF of Tchsc70-3 encoded a polypeptide of 663 amino acids with a predicted molecular weight of 73.34 kDa and a pI of 5.12.
The two TcHSP70 sequences contained three conserved signatures, an ATP-GTP binding site and a non-organellar consensus motif (Fig 4). In addition, the KDEL motif was identified in the deduced amino acid sequence of Tchsc70-3. The EEVD motif was found at the C-terminus of TcHSP70.
Tchsp90. The full-length cDNA of Tchsp90 was 2643 bp, including an ORF of 2181 bp, a 5'-UTR of 145 bp and a 3'-UTR of 317 bp (GenBank accession number MH490980). The ORF of Tchsp90 encoded a polypeptide of 726 amino acids with a predicted molecular weight of 83.48 kDa and a pI of 4.88.
Phylogenetic analysis of TcHsps. Phylogenetic trees were constructed based on the deduced amino acid sequences of Tchsps and their homologous sequences by the neighborjoining method. The results revealed that HSP10 sequences from T. chilonis and T. pretiosum   were clustered together into a single branch (Fig 6(A)). Two HSP60 sequences of endoparasitoid wasps (T. chilonis and P. puparum) were clustered within a branch (Fig 6(C)). A similar result was also found in the phylogenetic tree constructed with HSP90 sequences (Fig 6(E)). TcHSP21.6 showed a high similarity with HSP21.5 and HSP21.4 from other insects (Fig 6(B)). These sHSPs were clustered together and were separated from HSP beta-1 sequences. The sequences of the HSP70 family from insects presented two clusters, one with HSP70 sequences and another with HSC70-3 sequences (Fig 6(D)). The two TcHSP70 sequences showed a close relationship with their homologous sequences from the hymenopteran species.  6 and Tchsp60 decreased from the larval stage to the pupal stage and was maintained at a low level during the pupal and adult stages (Fig 7). Similarly, Tchsc70-3 had the highest expression level during the larval stage and the lowest level during the pupal stage, while it was upregulated from the pupal stage to the adult stage. In contrast, the expression of Tchsp70 and Tchsp90 peaked in the adult stage, with 5.63-and 3.27-fold increases, respectively compared to the levels in the larval stage.

Discussion
In this study, full-length cDNAs of six HSP genes were obtained from T. chilonis, including Tchsp10, Tchsp21.6, Tchsp60, Tchsp70, Tchsc70-3 and Tchsp90. TcHSP10 contained a mobile Cloning and expression of six hsps in T. chilonis loop, which is consistent with the characteristic of other HSP10 sequences described in many studies [31][32][33]. Through the mobile loop, HSP10 interacts with the HSP60, which helps the folding of protein [34,35]. Tchsp21.6 encoded a polypeptide of 191 amino acids with a predicted molecular weight of 21.68 kDa. The deduced amino acid sequence of Tchsp21.6 contained an α-crystallin domain (ACD), which is a characteristic feature of the sHSPs family [36,37]. This family is composed of many members containing variable N-and C-terminal extensions [32,38]. TcHSP60 belongs to the mitochondrial HSP60 family, containing a conserved signature motif (AAVEEGIVPGGG) and a GGM motif [23,39]. The GGM motif at the C-terminus has been suggested to provide a suitable physical environment for protein folding [40]. The ATP/ADP binding sites were found in TcHSP60, which have also been identified in HSP60 sequences from Rhopalosiphum padi (L.) (Homoptera: Aphididae) and Lucilia cuprina (Diptera: Calliphoridae) [39,41]. The highly conserved motif among HSP60 sequences may indicate that a similar mechanism of coupling ATP hydrolysis to the substrate-refolding process exists [14,39,42]. The two TcHSP70 sequences had three conserved HSP70 family signatures and a non-organellar consensus motif, in accordance with the structures of the HSP70 sequences described in Nilaparvata lugens (Homoptera: Delphacidae), Sitodiplosis mosellana (Diptera: Cecidomyiidae) and Habrobracon hebetor (Hymenoptera: Braconidae) [43][44][45]. The two TcHSP70 sequences showed high similarity with their homologous sequences from other insects. These results indicated that the members of HSP70 family are highly conserved [46].
It has been well reported that HSPs are involved in the development of insects [47,48]. However, the expression patterns of hsps during development vary in insects [49,50]. For example, the expression of hsp60 increases from the larval stage to the adult stage in Liriomyza sativa (Diptera: Agromyzidae), while it decreases from nymph to adult in R. padi [39,47]. In this study, Tchsp10 and Tchsp60 levels decreased during development, which is consistent with the findings of report on Galeruca daurica (Coleoptera: Chrysomelidae) [50]. Moreover, Tchsp21.6 showed the same expression pattern as Tchsp10 and Tchsp60. The high expression of three Tchsps in the larval stage indicated that these genes may be related to larval development. In T. chilonis, Tchsp70 and Tchsp90 levels peaked at the adult stage, and Tchsc70-3 expression was upregulated from the pupal stage to the adult stage. It is different from the findings in S. exigua and Frankliniella occidentalis (Thysanoptera: Thripidae) [48,49]. In contrast, in the endoparasitoid wasp M. cingulum, hsp70 and hsp90 are highly expressed in the pupal and adult stages [26]. The high expression of hsp70 begins at the third-instar larval stage, when C. vestalis comes out of the host [27]. As an egg endoparasitoid, adults of T. chilonis emerge from host, thus facing very different environmental stresses. These HSP genes, i.e., Tchsp70, Tchsp90 and Tchsc70-3, might be needed to overcome these challenges.
HSPs also play important roles in the response to temperature stress [51]. As described in the introduction, HSPs are molecular chaperones that help to prevent potential damage to cellular and molecular structures under temperature and other stresses [15,52]. Altered expression patterns of hsps have been widely reported under temperature stresses, although these responses seem to be species-specific among insects [39,43]. In T. pui, the expression of hsp90, rather than hsp70, changes in response to temperature [22]. However, hsp90 and hsp70 in Empoasca onukii (Hemiptera: Cicadellidae) are both highly expressed under cold and heat treatments [53]. Cchsp60 in C. chilonis responds to cold stress but is insensitive to heat stress [24]. In contrast, the highest expression level of hsp60 in P. puparum appears at 36˚C [23]. In this study, Tchsps were sensitive to high temperatures (32 and 40˚C), which is consistent with the expression patterns of hsps observed in other species [23,48,50]. The expression of Tchsp10, Tchsp21. 6 and Tchsp60 showed the same expression pattern in response to high temperatures, each being significantly upregulated under high temperatures but with no significant differences at 32 and 40˚C. The upregulation of hsp10 and hsp60 has also been reported in Apostichopus japonicus (Echinodermata: Holothuroidea) and G. daurica [31,50]. HSP10 is considered the co-chaperone of HSP60 and interacts with HSP60 in the same heat shock pathway [33,54]. TcHSP21.6 belongs to the sHSPs family and showed high similarity with HSP21.5 and HSP21.4, which have been proven to respond to heat stress in E. pela female adults and in B. mori [55,56]. Tchsp70 exhibited the highest expression at 40˚C. TcHSC70-3 is a constitutively expressed protein that is also induced by heat. These results agreed with the reports that the HSP70 family includes the major heat shock proteins induced by thermal stresses [24,26,48]. Tchsp90 exhibited the same expression pattern as the two TcHSP70 genes, being significantly upregulated from 32 to 40˚C. Similar response patterns have been reported in E. onukii, S. exigua and two Liriomyza species [21,48,53]. The upregulation of Tchsps expression in T. chilonis may contribute to the improvement of thermal tolerance. Some reports have documented that pupae of T. chilonis can endure a high temperature of 40˚C [57,58]. On the other hand, the heat-responsive expression of Tchsps also implies that the expression of Tchsps could be a potential indicator of the heat stress response [8,16]. Further study is needed to support this speculation. Cold treatments (10 and 17˚C) for 1 h led to a slight, but not significant, increase in the expression of all Tchsps, indicating that Tchsps are insensitive to low temperatures. In different insects, the expression patterns of these genes vary under cold exposure. For instance, hsp60 cannot be induced by cold in two Liriomyza species but could be upregulated in response to low temperature in C. chilonis [21,24]. The expression of hsp70 decreases in response to cold shock in N. lugens while it increases in E. onukii [43,53]. Some reports have indicated that the recovery from cold, rather than the direct cold stress, triggers the high expression of HSP genes [49,59].
Under the 40˚C treatment, the expression of all Tchsps exhibited a time-dependent response. Except for the expression level of Tchsp21.6 at 8 h, expression levels of these genes significantly increased after 1 h of exposure to 40˚C and dramatically decreased at subsequent time points. This result indicated that the induction of hsps may be rapid and transitory. This phenomenon has also been reported in numerous studies [21,31,44]. Many studies have attributed the decrease in hsp expression to the energy balance and metabolic disorder [50]. In addition, previous studies have speculated that the synthesis of HSPs requires excess energy consumption, imposing stresses on various metabolic activities [60,61]. The activity of enzymes is restricted under long-term heat exposure, which may also result in the decrease of hsp expression [62]. Under the 10˚C treatment, the expression of Tchsp21.6, Tchsp60, Tchsc70-3 and Tchsp90 did not dramatically change at any time points. Tchsp10 and Tchsp70 exhibited a low-intensity cold response at 4 and 8 h. This result suggested that Tchsps have low or no sensitivity to cold temperatures.

Conclusions
In summary, six Tchsps were cloned and characterized from T. chilonis, namely, Tchsp10, Tchsp21.6, Tchsp60, Tchsp70, Tchsc70-3 and Tchsp90. These Tchsps exhibited different expression profiles at different developmental stages, suggesting they may be involved in the development of T. chilonis. In pupae of T. chilonis, the expression profiles of these genes could be induced by heat shocks (32 and 40˚C for 1 h) but did not change in response to cold shocks (10 and 17˚C for 1 h). In addition, their expression levels showed time-dependent responses to heat exposure. Tchsp10 and Tchsp70 exhibited a low-intensity cold response at 4 and 8 h. However, Tchsp21.6, Tchsp60, Tchsc70-3 and Tchsp90 did not respond to cold exposure. Due to the difficultly of sampling caused by the tiny size and parasitic characteristics of T. chilonis, our study initially explored the expression patterns of hsps during development and temperature stresses. Our study may aid in a better understanding of the roles of hsps at different development stages and in response to temperature stresses.
Supporting information S1