Characterization and Expression of Genes Encoding Three Small Heat Shock Proteins in Sesamia inferens (Lepidoptera: Noctuidae)

The pink stem borer, Sesamia inferens (Walker), is a major pest of rice and is endemic in China and other parts of Asia. Small heat shock proteins (sHSPs) encompass a diverse, widespread class of stress proteins that have not been characterized in S. inferens. In the present study, we isolated and characterized three S. inferens genes that encode members of the α-crystallin/sHSP family, namely, Sihsp21.4, Sihsp20.6, and Sihsp19.6. The three cDNAs encoded proteins of 187, 183 and 174 amino acids with calculated molecular weights of 21.4, 20.6 and 19.6 kDa, respectively. The deduced amino acid sequences of the three genes showed strong similarity to sHSPs identified in other lepidopteran insects. Sihsp21.4 contained an intron, but Sihsp20.6 and Sihsp19.6 lacked introns. Real-time quantitative PCR analyses revealed that Sihsp21.4 was most strongly expressed in S. inferens heads; Whereas expression of Sihsp20.6 and Sihsp19.6 was highest in eggs. The three S. inferens sHSP genes were up-regulated during low temperature stress. In summary, our results show that S. inferens sHSP genes have distinct regulatory roles in the physiology of S. inferens.

The pink stem borer, Sesamia inferens (Walker) (Lepidoptera: Noctuidae), is a major pest of rice in China and other parts of Asia, and recently, damage incited by S. inferens has become more serious [22]. According to our previous surveys, this pest now occurs in the more northern regions of China. Many studies of S. inferens have focused on biological characteristics [23][24][25][26][27][28]. We previously demonstrated that S. inferens still survived during exposure to low temperatures [29].
The underlying mechanisms that explain sudden outbreaks and the widespread distribution of S. inferens remain obscure. Hence, expression analysis of relevant genes, such as those encoding sHSPs, may provide insight on the incidence of S. inferens. To investigate whether shsps expression regulates cold tolerance in S. inferens, we cloned three genes encoding sHSPs from this insect pest. The structure of these genes was examined, and we analyzed their expression in different tissues and stages of insect development. Our results indicate that expression of the three shsps is modulated in response to cold stress.

Phylogenetic Analysis of S. inferens sHSPs
The deduced amino acid sequences of the three shsps displayed a high degree of relatedness with orthologous proteins reported in other insects. To compare S. inferens sHSPs with those from other insects, ClustalX and MEGA 6.06 were used to perform multiple phylogenetic analyses, including neighbor-joining, minimum evolution, maximum likelihood, and maximum parsiomony. The four resulting phylogenetic trees were similar; Thus, only the neighbor-joining tree is shown (Figure 3). The tree could be divided into two major clusters; S. inferens HSP19.6 and HSP20.6, which show high sequence similarity, were grouped together in a well-supported cluster ( Figure 3). S. inferens HSP19.6 showed 97% amino acid identity with Sesamia nonagrioides HSP19.5, and S. inferens HSP20.6 exhibited 98% identity with S. nonagrioides HSP20.8 ( Figure 3

Expression of Genes Encoding sHSPs in S. inferens Tissues
qRT-PCR was used to study the expression profiles of the three shsps in S. inferens. The presence of single, sharply defined peaks in melting curve analysis of the three shsps and six reference genes was confirmed. A standard curve was generated for each gene using eight ten-fold serial dilutions (1×, 10×, 10 2 ×, 10 3 ×, 10 4 ×, 10 5 ×, 10 6 × and 10 7 ×) of the pooled cDNAs. The PCR efficiency (as calculated from the standard curve) and correlation coefficient (R 2 ) for each standard curve are shown in Table 2, and the parameters satisfied the basic requirements for quantitative real-time PCR [32].

Expression of Genes Encoding sHSPs in Different Developmental Stages
The three S. inferens sHSP genes were expressed in all stages of S. inferens development, although the expression levels varied widely. Expression of Sihsp21.4 was highest in female adults ( Figure 5B2) and lowest in third instar larvae (F10,22 = 3.027, p = 0.015). Expression of Sihsp20.6 and Sihsp19.6 was highest in eggs, and this difference was significant when compared to expression in other developmental stages (Sihsp20.

Discussion
Overwintering insects are exposed to temperature stress in nature and must adopt specialized adaptive mechanisms to survive low temperatures. The induction of small heat shock proteins is a potential survival mechanism during temperature stress [19]; However, to our knowledge, the study of sHSPs in S. inferens has not been previously undertaken. In the present study, we cloned three members of the sHSP family from S. inferens. Analysis of the cDNA sequences and deduced ORFs indicated that Sihsp21.4, Sihsp20.6, and Sihsp19.6 encoded proteins containing 187, 183, and 174 amino acids, respectively. The predicted amino acid sequences shared considerable sequence similarity with sHSP from other insects and α-crystallin proteins from vertebrate eye lenses. Phylogenetic analysis indicated that S. inferens HSP19.6 and S. nonagrioides HSP19.5 clustered with the same group; Whereas S. inferens HSP20.6 and S. nonagrioides HSP20.8 clustered together in another group (Figure 3). S. inferens HSP21.4 and five other lepidopteran HSP21.4 orthologs grouped together in a well-supported cluster, which supports the accuracy of the sequence analysis conducted in the present study. S. inferens HSP19.6 and HSP20.6, which show high sequence similarity, grouped together in a well-supported cluster, whereas S. inferens HSP21.4 sorted to a different cluster. Thus, Sihsp21.4 may have evolved differently from Sihsp20.6 and Sihsp19.6. It is interesting to note that sHSP orthologs from S. litura and C. suppressalis show phylogenetic similarities to the S. inferens proteins identified in the present study [19,33].
A negative correlation between intron size and the level of gene expression has been suggested previously; In other words, genes containing smaller introns or lacking introns were more highly expressed than genes containing large or multiple introns [34]. It is also possible that genes either lacking or containing shorter introns may be more sensitive to environmental stresses. Based on chromosomal location and intron number, sHSP genes could be subdivided into two types: Orthologous or species-specific [35]. Thus, the three shsps from S. inferens could be classified into two groups: Orthologous that contained introns (Sihsp21.4) and species-specific forms lacking introns (Sihsp20.6 and Sihsp19.6).
The various functions of small heat shock proteins in insect tissues are not well-understood. One possibility is that sHSPs play important, specialized roles in maintaining normal functioning in different tissues [15]. In this study, Sihsp21.4 was highly expressed in S. inferens heads, which is similar to the high expression of hsp19.1 and hsp22.6 reported in B. mori heads [35]. The primary nerve center in insects, e.g., the supraoesophageal ganglion and suboesophageal ganglion, is located within the head; Thus, it is possible that sHSPs could protect the nerve centers from external injury. However, shsps of S. litura, Apis cerana cerana, and C. suppressalis were expressed at very high levels in malpighian tubules and hindgut tissues [19,33,36]. Malpighian tubules and hindguts function by reabsorbing water, salts, and other substances prior to excretion by the insect; Thus, it remains unclear why shsps were highly expressed in these tissues. One hypothesis is that sHSPs protect these tissues from potentially toxic substances [19]. Taken together, our data suggest that different shsps play distinct roles in the physiology of S. inferens.
sHSPs play important roles in development, including the regulation of insect development [33,37,38]. For instance, l2efl, a type of shsp, reached a maximum level of expression in the third instar larvae of D. melanogaster [39]. However, in Lucilia cuprina, expression of hsp24 was lowest in third instar larvae [37]. Lu et al. reported a high level of Cshsp21.7a expression in first instar larvae of C. suppressalis, whereas the highest expression of Cshsp19. 8,Cshsp21.4,Cshsp21.5,and Cshsp21.7b was observed in C. suppressalis adults; In this study, expression of Sihsp20.6 and Sihsp19.6 was highest in insect eggs, whereas Sihsp21.4 expression was highest in female adults [19]. Thus our data support the hypothesis that sHSPs have evolved specific roles in different stages of insect development.
More in-depth studies are needed to clarify the role of sHSPs in insect behavior and development. Future investigations will help reveal the underlying physiological mechanisms of shsps in S. inferens, thus enhancing our ability to implement more effective control measures for this significant pest.

Insects
Populations of S. inferens were collected from a suburb of Yangzhou (32°39'N, 119°42'E), located in the Jiangsu province. The pink stem borers were reared for more than 3 generations in environmental chambers maintained at 27 ± 1 °C with a 16:8 (light/dark) photoperiod and 60%-70% relative humidity as described previously [42].

Reverse Transcription Polymerase Chain Reaction (PCR) and Rapid-Amplification of cDNA Ends (RACE)
Total RNA was extracted from S. inferens using the SV Total RNA isolation system (Promega, Madison, WI, USA) and then treated with DNase I. The integrity of RNA was verified by comparing RNA bands in gels stained with ethidium bromide. RNA purity was analyzed at 260 and 280 nm using a spectrophotometer (Eppendorf BioPhotometer plus, Eppendorf, Germany). cDNA copies of genes encoding sHSPs were synthesized using oligo(dT)18 primers (Fermentas, Helsingborg, Sweden). Degenerate primers for PCR were designed using consensus sequences of shsps obtained from several lepidopteran insects; These sequences were previously deposited in GenBank (Table 1). Degenerate primers of Sihsp21.4 were designed using consensus sequences of H. armigera hsp21.4, Spodoptera litura hsp21.4 and C. suppressalis hsp21.4; Degenerate primers of Sihsp20.6 were designed using consensus sequences of S. nonagrioides hsp20.8, S. litura hsp20.4 and B. mori hsp20.4; Degenerate primers of Sihsp19.6 were designed using consensus sequences of S. nonagrioides hsp19.5, B. mori hsp19.5 and Plutella xylostella hsp19.5. And the amino acid regions used to design degenerate primers are conserved regions of each gene. Full-length cDNAs were obtained using 5' and 3' RACE (SMARTer™ RACE, Clontech, Palo Alto, CA, USA). Primers for RACE were designed based on partial sequence information derived from shsps cDNA fragments (Table 1). Complete sequences of intact ORFs were confirmed by 5' RACE cDNA. Products were purified using the AxyPrep™ DNA Gel Extraction Kit (Axygen, Union City, CA, USA), cloned into pGEM-T Easy Vector (Promega), and then transformed into Escherichia coli DH5α cells for subsequent sequence analysis.

Characterization of Genomic DNA
The genomic DNA of S. inferens was extracted using the AxyPrep™ Multisource Genomic DNA Kit (Axygen Biosciences, Union City, CA, USA). Specific primer pairs (Table 1) were designed to amplify genomic fragments based on analysis of full-length cDNAs. The products were purified using the AxyPrep™ DNA Gel Extraction Kit (Axygen), cloned into pGEM-T EasyVector (Promega), and transformed into E. coli DH5α for sequence analysis.

Tissues Samples
The larvae selected for analysis were similar in size and randomly assigned to experimental groups. Each group contained ten larvae, and each experiment was repeated three times. Larvae were anesthetized on ice prior to dissection. The head, epidermis, fat body, foregut, midgut, hindgut, malpighian tubules, haemocytes, and salivary glands were collected from larvae and rinsed with a 0.9% sodium chloride solution. The samples were frozen immediately in liquid nitrogen and stored at −70 °C prior to real-time PCR analyses.

Samples Representing Developmental Stages and Sex
Samples included egg masses, the first, second, third, fourth, fifth and sixth instar larvae, male and female pupae, and one-day-old male and female adults; Samples were randomly selected for the experiment. The samples were frozen immediately in liquid nitrogen and stored at −70 °C until needed for analyses.

Cold Tolerance Samples
In this experiment, larvae representing the fifth instar were placed individually in glass tubes, and groups of ten were then exposed to various temperatures (−8, −6, −4, −2 and 0 °C) for 2 h in a constant-temperature incubator (DC-3010, Jiangnan Equipment, Changzhou, China). The larvae were recovered at 27 ± 1 °C for 2 h, after which surviving larvae were frozen in liquid nitrogen and stored at −70 °C. A set of larvae maintained at 27 ± 1 °C was regarded as a control group. Each treatment included at least three surviving larvae.

Quantitative Real-Time PCR
Total RNA was extracted using the methods described above for reverse transcription PCR and RACE. RNA (0.5 μg) was reverse-transcribed into first-strand cDNA using the Bio-Rad iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Real-time PCR reactions were performed in a 20 μL reaction volume comprised of 10 μL Bio-Rad iTaq™ Universal SYBR ® Green supermix (Bio-Rad, 2×), 1 μL of each gene-specific primer (10 μM) ( Table 2), 2 μL of cDNA template, and 6 μL of ddH2O. Reactions were carried out using a CFX-96 real-time PCR system (Bio-Rad) under the following conditions: 3 min at 95 °C, 40 cycles of denaturation at 95 °C for 30 s, and annealing at the Tm for each gene (30 s; Table 2). Each treatment included three replicates, and each reaction was run in triplicate.

Data Analysis
ORFs were identified using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The deduced amino acid sequences were aligned using CLUSTAL X1.83 [43]. Sequence analysis tools of the ExPASy Molecular Biology Server (Swiss Institute of Bioinformatics, Basel, Switzerland) were used to analyze the deduced sHSP sequences, including Translate, Compute pI/MW, and Blast. Amino acid sequences were used to estimate phylogeny using neighbor-joining, minimum evolution, maximum likelihood, and maximum parsimony methods. Phylogenetic trees were constructed with 1000 bootstrap replicates using MEGA version 6.06 (Tempe, AZ, USA) [44].
Homology models were generated using Protein Homology/analogy Recognition Engine software version 2.0 (http://www.sbg.bio.ic.ac.uk/~phyre2/html) [45]. The Chimera tool was used to visualize the 3D coordinates for the atoms in the predicted protein models [46]. qRT-PCR data were analyzed using the Bio-Rad CFX Manager™ 3.1 software (Bio-Rad). The threshold cycle (Ct value) denotes the cycle at which the fluorescent signal first shows significant difference with respect to the background. All biological replicates were used to calculate the average Ct values. Relative expressions were calculated using the 2 −ΔΔCt method [47]. Three genes (RPS13, RPS20 and EF1) were used for normalizing gene expression in different tissues. 18S rRNA, EF1 and GAPDH were used as reference genes in different developmental stages and sexes. 18S rRNA, RPS20 and TUB were used for normalizing gene expression at different temperatures. The means of the reference genes were used as normalization under different experimental conditions [48]. The above-mentioned reference genes were previously validated in a research study that has been submitted elsewhere. Tukey's test was conducted for statistical analysis using PASW Statistics 18.0 (SPSS Inc., Chicago, IL, USA).

Conclusions
In conclusion, we cloned three genes encoding sHSPs from S. inferens. The structure of these genes was examined, and we analyzed their expression in different tissues and stages of insect development. Our results also indicate that expression of the three shsps is modulated in response to cold stress.