Different Responses of Cytoplasmic and Endoplasmic Reticulum Hsp90 Genes from Eogystia hippophaecola (Lepidoptera: Cossidae) to Cold Stress

Eogystia hippophaecola Hua, Chou, Fang et Chen (Lepidoptera: Cossidae) is an important borer pest of the sea buckthorn forest (Hippophae rhamnoides L.) in China. Its larvae, which are highly cold tolerant, mainly overwinter in sea buckthorn roots. Heat shock proteins (Hsps) are important molecular chaperones that have been linked to cold tolerance in insects. In this study, we cloned the open reading frames (ORFs) of two Hsp90 genes from E. hippophaecola, EhHsp90-1 and EhHsp90-2, and analyzed their expression under cold stress by qRT-PCR. EhHsp90-1 and EhHsp90-2 are 2154 and 2346 bp in length, respectively, encoding 717 and 781 amino acids. The deduced amino acid sequences contain the conserved signature sequences of the Hsp90 family and the C-terminus characteristic sequence of cytoplasmic or endoplasmic reticulum Hsp90 protein. Phylogenetic analysis revealed the amino acid sequences of EhHsp90-1 and EhHsp90-2 were very similar to the corresponding proteins from Lepidoptera. Under various low-temperature treatments lasting 2 h, EhHsp90-1 and EhHsp90-2 exhibited similar expression patterns, increasing first and then decreasing. At −5 °C, EhHsp90-1 was significantly up-regulated after 12 h, whereas EhHsp90-2 was up-regulated after just 1 h and reached its highest level at 2 h; however, the overall degree of upregulation was greater for EhHsp90-1. Subsequently, the expression level of EhHsp90-2 fluctuated with time. Our results suggest that the two Hsp90s play important roles in E. hippophaecola larvae response to cold stress, but that their response times and the magnitudes of their responses to low-temperature stress differed significantly, providing a theoretical basis for further studying the molecular mechanism of cold tolerance in E. hippophaecola larvae.


Introduction
Insects are poikilotherms, i.e., their body temperatures are susceptible to environmental conditions. Accordingly, environmental temperature is one of the most important factors affecting insects' many biological processes. Previous studies show that in addition to insects' genetic basis, insects also adopt different physiological and biochemical strategies to respond to low-temperature stress [1]. Among these strategies, there are many genes and their encoded protein products associate with tolerance to low temperatures in insects, including heat shock proteins (Hsps) [2,3], antifreeze proteins [4], trehalose synthase [5], chitinase [6], ice-nucleating protein [7], and the DCA [8] and FST proteins [9]. The induction of heat shock proteins is one of the most extensively studied areas, and is thought to be a key determinant to improve insect cold tolerance.

Cold Stress Treatments
The 13-15 instar larvae were randomly selected for the experiment and each larva was placed in a 50 ml perforated centrifuge tube, then they were immediately transferred to different cold temperatures from the common culturing temperature. Treatments at different temperatures were performed as follows: 13-15 instar larvae were treated at five temperatures (25 • C, 5 • C, 0 • C, −5 • C, and −10 • C) for 2 h, and larvae treated at 25 • C were regarded as the control group. Treatments for different times were performed as follows: 13-15 instar larvae were placed at −5 • C for 1, 2, 6, 12, or 24 h; larvae treated for 0 h were used as the control group. Each treatment was repeated three times. The treated larvae were immediately frozen in liquid nitrogen and placed in a −80 • C refrigerator for later use.

Isolation of Total RNA and Synthesis of First-Strand cDNA
Total RNA from treated larvae were isolated using the TRIzol reagent (number 15596018; Invitrogen, Carlsbad, CA, USA) and the RNeasy Plus Mini Kit (number 74134; Qiagen, Hilden, Germany), and then stored at −80 • C. The concentration, purity, and quantity of the RNA were assessed by agarose gel electrophoresis and the NanoDrop 8000 (Thermo Scientific, Waltham, MA, USA). First-strand cDNA was synthesized from 1 µg of total RNA using the PrimeScript RT Reagent Kit (number RR047Q; TaKaRa, Dalian, China), and then stored at −20 • C until use.

Cloning of EhHsp90 ORFs
Based on the larval transcriptome data established previously in our laboratory [41], we screened Hsp90 homologs and predicted the ORF regions using an ORF finder (https://www.ncbi.nlm.nih.gov/ orffinder, 22 April 2019). The complete ORFs were named EhHsp90-1 and EhHsp90-2. Primers were designed against two full-length sequences using Primer Premier 5.0, and the sequences of all primers used in this study are summarized in supplementary material 1. PCR reactions were conducted as follows: 35 cycles of 98 • C for 10 s, 53 • C for 15 s (EhHsp90-1) or 55 • C for 5 s (EhHsp90-2), 72 • C for 5 s, and 1 min at 72 • C.
The amplified PCR products were confirmed in a 1.0% agarose gel (Oxoid, Hants, UK)and then the purified PCR products were cloned into the pEASY-Blunt Simple Cloning Vector (number CB111-01; TransGen Biotech, Beijing, China). The ligated product was transformed into Trans1-T1 Phage Resistant Chemically Competent Cells (number CD501-01, TransGen Biotech, Beijing, China. Then, the positive colonies were identified using blue and white selection and sequenced at RuiBiotech Co., Ltd (Beijing, China). After alignment, the ORF sequences were obtained.

Expression of EhHsp90 under Different Treatments
Specific primers (supplementary material 1) for qRT-PCR were designed based on the sequences of ORFs using Primer Premier 5.0 [45]. The E. hippophaecola actin gene was used as a reference [40,41,46] and we used Bestkeeper to evaluate the std value (<1) of actin gene under different temperatures which indicated that actin gene was stable [47]. The qRT-PCR was carried out on a Bio-Rad CFX96 PCR machine (Hercules, CA) in a reaction mixture of 12.5 µL consisting of 6.25 µL of SYBR Premix Ex Taq II (2×) (Takara, Dalian, China), 0.5 µL of each primer, 1 µL of cDNA template, and 4.25 µL of ddH 2 O. The reactions were conducted as follows: 95 • C for 30 s, 40 cycles of 5 s at 95 • C, 30 s at 60 • C, and 15 s at 95 • C; the Ct value was read at the end of the reaction. Each qRT-PCR reaction was performed in three biological replicates, and each sample was technically repeated three times. The relative expression levels of Hsps were calculated by the 2 −∆∆Ct method [48]. The Ct mean and standard error of each treatment were calculated using the Bio-Rad CFX Manager (version 3.1.1517.0823) software [49], and the relative expression levels of EhHsp90 were statistically analyzed by one-way analysis of variance (ANOVA) and Tukey's test using spss 18.0 software (spss, Inc., Chicago, IL, USA). Differences were considered statistically significant at p < 0.05.

Characterization of EhHsp90
The ORFs of two Hsp90 clones were named EhHsp90-1 (GenBank accession number: MN565559) and EhHsp90-2 (GenBank accession number: MN565560). The gene sequences and their deduced amino acid sequences are shown in Figures 1 and 2, respectively. The sequence characteristics are shown in Table 1 The protein encoded by EhHsp90-1 is unstable, whereas the protein encoded by EhHsp90-2 is relatively stable; both are hydrophilic. In addition, the C-terminus of EhHsp90-1 has the common characteristic motif (EEVD) of all eukaryotic Hsp90 proteins, while the C-terminus of EhHsp90-2 has an ER retention sequence (HDEL). For EhHsp90-2, these is an extra~20aa with signal peptide that only exists in the N-terminal ( Figure 2). 60°C, and 15 s at 95°C; the Ct value was read at the end of the reaction. Each qRT-PCR reaction was performed in three biological replicates, and each sample was technically repeated three times. The relative expression levels of Hsps were calculated by the 2 −∆∆Ct method [48]. The Ct mean and standard error of each treatment were calculated using the Bio-Rad CFX Manager (version 3.1.1517.0823) software [49], and the relative expression levels of EhHsp90 were statistically analyzed by one-way analysis of variance (ANOVA) and Tukey's test using spss 18.0 software (spss, Inc., Chicago, IL, USA). Differences were considered statistically significant at p < 0.05.

Characterization of EhHsp90
The ORFs of two Hsp90 clones were named EhHsp90-1 (GenBank accession number: MN565559) and EhHsp90-2 (GenBank accession number: MN565560). The gene sequences and their deduced amino acid sequences are shown in Figure 1 and 2, respectively. The sequence characteristics are shown in Table 1 The protein encoded by EhHsp90-1 is unstable, whereas the protein encoded by EhHsp90-2 is relatively stable; both are hydrophilic. In addition, the C-terminus of EhHsp90-1 has the common characteristic motif (EEVD) of all eukaryotic Hsp90 proteins, while the C-terminus of EhHsp90-2 has an ER retention sequence (HDEL). For EhHsp90-2, these is an extra ~20aa with signal peptide that only exists in the N-terminal ( Figure 2).
Thirty-nine full-length hsp90 amino acid sequences from Lepidoptera, Diptera, Hemiptera, and Coleoptera were employed for these genetic analysis ( Figure 4). The phylogenetic tree used ML and NJ were distinctly divided into two groups: Cy-Hsp90s and ER-Hsp90s. Every group was further divided into four orders: Lepidoptera, Diptera, Hemiptera, and Coleoptera. E. hippophaecola Hsp90s belong to different classes of Hsp90, and cluster with Hsp90 from Lepidoptera. Moreover, branches of the same family insects cluster well in each order of insects.        I  I  I  I  I  i   I  I  I  I  I

Expression of EhHsp90 under Different Low-Temperature Treatments
The expression levels of the two Hsp90 after 2 h treatments at various low-temperature treatments are shown in Figure 5. The relative expression of EhHsp90-1 was the highest at 0 • C but did not differ significantly from the control; however, the expression at −10 • C was significantly lower than at −5 • C (p = 0.04). In addition, the relative expression of EhHsp90-2 was the highest (1.75-fold) at −5 • C and significantly higher than at 5 • C (p = 0.01), and there was also a significant decrease between −10 • C and −5 • C (p = 0.03). Thus, the levels of EhHsp90-1 and EhHsp90-2 first increased, and then decreased, with decreasing temperature. did not differ significantly from the control; however, the expression at −10 °C was significantly lower than at −5 °C (p = 0.04). In addition, the relative expression of EhHsp90-2 was the highest (1.75-fold) at −5 °C and significantly higher than at 5 ℃ (p = 0.01), and there was also a significant decrease between −10 °C and −5 °C (p = 0.03). Thus, the levels of EhHsp90-1 and EhHsp90-2 first increased, and then decreased, with decreasing temperature.

Expression of EhHsp90 under Different Treatment Times
The expression levels of the two Hsp90 in larvae exposed to −5 °C for various times are shown in Figure 6. Expression of EhHsp90-1 was the highest at −5 °C for 12 h, 3.44 times higher than in the control, and remained significantly higher at 24 h (p = 0.04). However, the expression pattern of EhHsp90-2 differed: the level of EhHsp90-2 was up-regulated after 1 h and reached a maximum at 2 h. But was significantly lower at 6 h than at 2 h (p = 0.01). After that, the expression level of EhHsp90-2 fluctuated with time. However, the overall degree of upregulation was greater for EhHsp90-1. Thus, under cold stress, the expression patterns of EhHsp90-1 and EhHsp90-2 differed over time.

Expression of EhHsp90 under Different Treatment Times
The expression levels of the two Hsp90 in larvae exposed to −5 • C for various times are shown in Figure 6. Expression of EhHsp90-1 was the highest at −5 • C for 12 h, 3.44 times higher than in the control, and remained significantly higher at 24 h (p = 0.04). However, the expression pattern of EhHsp90-2 differed: the level of EhHsp90-2 was up-regulated after 1 h and reached a maximum at 2 h. But was significantly lower at 6 h than at 2 h (p = 0.01). After that, the expression level of EhHsp90-2 fluctuated with time. However, the overall degree of upregulation was greater for EhHsp90-1. Thus, under cold stress, the expression patterns of EhHsp90-1 and EhHsp90-2 differed over time.

Characterization and Phylogenetic Analysis of EhHsp90
Environmental temperature is a key factor affecting the growth and development of insects. Hsps play important roles in various stress responses, especially in heat or cold adaptation in insects, and studies have shown that Hsps contribute to the improvement of insect cold tolerance [15,23,24]. In this study, we cloned two Hsp90 genes, EhHsp90-1 and EhHsp90-2, from the larvae of E. hippophaecola. Sequence analysis found both of the genes contain the characteristics motifs of the Hsp90 families. The presence of EEVD that binding to many cochaperones with small helical TRP domains at the EhHsp90-1 C-terminal indicates that EhHsp90-1 is a cytoplasmic Hsp [50][51][52], while the C-terminus of the EhHsp90-2 has a conserved HDEL motif which is an endoplasmic reticulum targeting sequence [28]. In addition, the ATPase domain was also found at the N-terminus of EhHsp90-1 and EhHsp90-2, indicating that Hsp90 is a highly conserved, ATP-regulated dimeric molecular chaperone. For EhHsp90-2, beside these domains, these is an extra ~20 aa with Gln-rich signal peptide that only exists in the N-terminal of endoplasmic reticulum Hsp90 not in that of cytoplasmic Hsp90 (Figure 2). Under the guidance of signal peptide, the newly synthesized protein enters the endoplasmic reticulum cavity, which also means that EhHsp90-2 is located differently from EhHsp90-1.
As expected, two E. hippophaecola hsp90s share high sequence similarities with their counterparts from other insects, and include conserved signature motifs that facilitate interaction with other proteins and enhance their chaperone function ( Figure 3) [52,53]. Phylogenetic analysis revealed that Cy-Hsp90s and ER-Hsp90s were clearly divided into two groups, and they evolved separately from a common ancestor but Cy-Hsp90 originated earlier than ER-Hsp90, indicating that these two molecular chaperones may be under differential selections during their evolution [28,54]. The EhHsp90-1 and EhHsp90-2 were in the different groups, and they clustered together with Hsp90 from Lepidoptera, respectively. However, because little is known about cold resistance genes and no Hsp sequence information is in GenBank from Cossidae insects, the EhHsp90s may be clustered with different family of Lepidoptera in ML and NJ methods. In addition, the two groups showed that Lepidoptera, Diptera, Hemiptera, and Coleoptera are were well segregates from each other, and even

Characterization and Phylogenetic Analysis of EhHsp90
Environmental temperature is a key factor affecting the growth and development of insects. Hsps play important roles in various stress responses, especially in heat or cold adaptation in insects, and studies have shown that Hsps contribute to the improvement of insect cold tolerance [15,23,24]. In this study, we cloned two Hsp90 genes, EhHsp90-1 and EhHsp90-2, from the larvae of E. hippophaecola. Sequence analysis found both of the genes contain the characteristics motifs of the Hsp90 families. The presence of EEVD that binding to many cochaperones with small helical TRP domains at the EhHsp90-1 C-terminal indicates that EhHsp90-1 is a cytoplasmic Hsp [50][51][52], while the C-terminus of the EhHsp90-2 has a conserved HDEL motif which is an endoplasmic reticulum targeting sequence [28]. In addition, the ATPase domain was also found at the N-terminus of EhHsp90-1 and EhHsp90-2, indicating that Hsp90 is a highly conserved, ATP-regulated dimeric molecular chaperone. For EhHsp90-2, beside these domains, these is an extra~20 aa with Gln-rich signal peptide that only exists in the N-terminal of endoplasmic reticulum Hsp90 not in that of cytoplasmic Hsp90 (Figure 2). Under the guidance of signal peptide, the newly synthesized protein enters the endoplasmic reticulum cavity, which also means that EhHsp90-2 is located differently from EhHsp90-1.
As expected, two E. hippophaecola hsp90s share high sequence similarities with their counterparts from other insects, and include conserved signature motifs that facilitate interaction with other proteins and enhance their chaperone function ( Figure 3) [52,53]. Phylogenetic analysis revealed that Cy-Hsp90s and ER-Hsp90s were clearly divided into two groups, and they evolved separately from a common ancestor but Cy-Hsp90 originated earlier than ER-Hsp90, indicating that these two molecular chaperones may be under differential selections during their evolution [28,54]. The EhHsp90-1 and EhHsp90-2 were in the different groups, and they clustered together with Hsp90 from Lepidoptera, respectively. However, because little is known about cold resistance genes and no Hsp sequence information is in GenBank from Cossidae insects, the EhHsp90s may be clustered with different family of Lepidoptera in ML and NJ methods. In addition, the two groups showed that Lepidoptera, Diptera, Hemiptera, and Coleoptera are were well segregates from each other, and even the different family were also well separated, supporting that Hsp90 protein allows good phylogenetic analysis [25,55].

Expression of EhHsp90 under Different Treatments
The 4-year-old larvae of E. hippophaecola are highly cold tolerant and understanding how E. hippophaecola responds to cold stress is crucially important to understand how they withstand extreme environmental temperatures [18]. In the Hsps families, up-regulation of Hsp90 gene plays an important role in resisting cold stress [56]. This study revealed the expression patterns of EhHsp90s under cold stress. When larvae were exposed to various cold temperatures for 2 h, expression levels of both EhHsp90-1 and EhHsp90-2 were up-regulated, indicating that EhHsp90s had a relationship with cold temperature changes and perhaps play important roles in surviving temperatures stress for E. hippophaecola larvae. In addition, EhHsp90-1 and EhHsp90-2 had similar expression patterns at different low temperatures, with the expression levels of both proteins reaching the highest point at −5 • C, however the expression level was significantly lower at −10 • C than at −5 • C. In general, the threshold temperature for Hsps induction is correlated with the typical temperatures at which species live [57] and Hsps are subjected to strict regulation by multiple molecular mechanisms [58]. Thus, it is possible that the ability of Hsp90s as molecular chaperones to completely mitigate the damage resulting from extreme cold stress and increase cold tolerance of insects are limited [18]. In our study, the downregulation of EhHsp90s at −10 • C below the supercooling point of −4 ± 0.5 • C of E. hippophaecola larvae [59] may not be induced or expressions of Hsp90s were affected by the regulator (HSF1 or σ32) [60]. Secondly, we observed no significant difference in the expression level of EhHsp90-1 treated at low temperature for 2 h, potentially because this did not provide sufficient time for induction. This was also reflected in experiments in which larvae were exposed to −5 • C for various times. Above 12 h, the expression level of EhHsp90-1 was significantly up-regulated. The observation that EhHsp90-1 is significantly expressed after long-term low-temperature stress is consistent with the highest expression of Hsp90 in M. separata after 12 h of low-temperature induction [61]. However, the temporal profile of EhHsp90-2 expression at −5 • C differed from that of EhHsp90-1. EhHsp90-2 was up-regulated after 1 h, reaching a maximum at 2 h, but the overall degree of upregulation was lower than that of EhHsp90-1. Subsequently, the expression level fluctuates with the extension of time. It may be related to the localization of the EhHsp90-2 in the ER. ER-Hsp90 is a widely expressed chaperone protein, also known as glucose-regulated protein (GRP94). When the body is consuming a great deal of energy, expression of ER-Hsp90 is rapidly induced [62]. Hence, EhHsp90-2 may exhibit short-term stress-dependent expression during larval adaptation to low temperatures, which consumes large amounts of energy. In addition, the expression levels of EhHsp90-1 and EhHsp90-2 were down-regulated after 24 h of low-temperature stress relative to 12 h. With the extension of the low temperature time, the accumulation of Hsp90 continues, however the large amount of expression of Hsp90 requires energy substances. At this time, insects down-regulate Hsp90 in order to maintain the basic biological processes, but the expression level is still higher than that of Hsp90 under normal [63]. Therefore, the EhHsp90s plays an important role in response to cold stress. Our comprehension of Hsp90s in response to low temperatures stress has a reference for understanding the capability of E. hippophaecola larvae to cold tolerance. However, further experiments are needed to explore the details of the mechanisms of Hsp90 genes in cold tolerance of E. hippophaecola larvae.

Conclusions
In this study, we successfully cloned two Hsp90 genes of E. hippophaecola for the first time, which contain the conserved signature sequences of the Hsp90 family. The phylogenetic analysis showed that proteins encoded by EhHsp90-1 and EhHsp90-2 are cytoplasmic and endoplasmic reticulum Hsp90 proteins, respectively, and that their amino acid sequences were very similar to the corresponding proteins from Lepidoptera. The expression patterns of EhHsp90-1 and EhHsp90-2 under cold stress showed that their response times and the magnitudes of their responses to low-temperature stress differed significantly. These results suggest that the two Hsp90s play important roles in E. hippophaecola larvae response to cold stress, and provide a theoretical basis for further studying the molecular mechanism of cold tolerance in E. hippophaecola larvae.