Differential expression patterns of two delta‐9‐acyl‐CoA desaturases in Thitarodes pui (Lepidoptera: Hepialidae) during different seasons and cold exposure

Abstract Thitarodes pui larvae have a limited distribution in the Tibetan Plateau and are the host of a parasitic fungus, Ophiocordyceps sinensis. Low temperature is a main environmental stress. However, understanding of T. pui cold adaptation mechanisms is insufficient. Delta‐9‐acyl‐CoA desaturase (D9D) is closely correlated with cold adaptation for many organisms. To further understand the cold adaptation processes in T. pui larvae, two D9Ds, TpdesatA and TpdesatB were sequenced, and expression patterns were investigated during different seasons and cold exposure (under 0°C) in the laboratory. The full lengths of two cDNAs are 1,290 bp and 1,603 bp, and the ORFs encode a polypeptide of 348 and 359 amino acids, respectively. Four transmembrane domains, three conserved histidine residues and five hydrophobic regions exist in these two sequences. The expression level of TpdesatA is up‐regulated in the long‐term cold exposure and negatively correlated with temperature in seasonal patterns. TpdesatB responds to cold temperature in short‐term cold exposure and positively corresponds temporarily in seasonal expression. Two D9Ds may have different substrate specificities, TpdesatA tends to use C16:0 and C18:0 as substrate while TpdesatB prefers C18:0. In conclusion, TpdesatA may play a very important role in T. pui cold tolerance and TpdesatB regulates function in short‐term cold exposure and content change of fatty acids in the body.


| INTRODUCTION
For overwintering insects, low temperature is one of the most serious environmental stresses affecting their survival. In fact, overwintering insects survive low temperature due to a variety of physiological and biochemical adaptations (Baust & Rojas, 1985;Clark et al., 2009). Until recently, cold hardiness in insects was most often discussed in terms of cryoprotectants, membrane lipids, and heat-shock proteins (Storey & Storey, 2012;Teets & Denlinger, 2013;Yocum, 2001). The transition of cell membrane lipids from a liquid crystalline phase to a gel phase is an important cause of cold injuries under nonfreezing conditions (Michaud & Denlinger, 2006). Further investigations revealed that accumulation of unsaturated fatty acids (UFAs) contributed to the fluidity of cellar membranes which are susceptible to cold (Khani, Moharamipour, & Barzegar, 2007;Koštá, Berkova, & Šimek, 2003;Michaud & Denlinger, 2006). In most cases, more UFAs and less saturated fatty acids (SFAs) were detected in response to cold exposure (Los & Murata, 2004;. Studies on the composition of cell membrane lipids in many species of microorganisms, plants, and animals under different temperatures have revealed the universal occurrence of remodeling of cell membrane lipids in response to changes in ambient temperature, a phenomenon known as homeoviscous adaptation (Hazel, 1995;Teets & Denlinger, 2013). The increasing UFAs are considered to play a role in maintaining the liquid crystalline phase at low temperatures (Kayukawa, Chen, Hoshizaki, & Ishikawa, 2007).
Delta-9-acyl-CoA desaturase (D9D) is an important enzyme that introduces a double bond into SFAs, and has been shown to play an essential role in cold hardiness by increasing the ratio of UFAs to SFAs in cell membranes (Hsieh & Kuo, 2005;Tiku, Gracey, Macartney, Beynon, & Cossins, 1996). Certain groups of reports demonstrated that up-regulation of the D9D gene occurs during cold exposure. Tiku et al. (1996) indicated that transcription of the D9D gene increased tenfold in the liver of cold-exposed carp. Similar findings were also proved in Oreochromis niloticus (Zerai, Fitzsimmons, & Collier, 2010), as well as Chanos chanos and Ctenopharyngodon idella (Hsieh & Kuo, 2005). For insects, the D9D gene was firstly proved to participate in cold adaptation mechanisms in Delia antique. In that study, twofold to tenfold up-regulation of the D9D gene was induced in brain tissues, malpighian tubules, and the midgut when D. antique was exposed to cold (Kayukawa et al., 2007). This same result occurred in Sarcophaga crassipalpis (Rinehart, Robich, & Denlinger, 2010), Folsomia candida (Waagner, Holmstrup, Bayley, & Jesper, 2013), and Aedes albopictus (Reynolds et al. 2012). Meanwhile, another kind of D9D, which was associated with dietary alterations, was found in Cyprinus carpio and Acheta domesticus (Batcabe, Howell, Blomquist, & Borgeson, 2000;Polley et al., 2003).
Our previous work indicated that proteins, total sugar, and total fat in the hemolymph of T. pui larvae showed negative correlation with soil temperature (Yi, Zhang, Guo, Min, & Zou, 2015). In addition, HSP90 of T. pui, rather than HSP70, responds to temperature changes and potentially plays a key role in cold tolerance (Zou, Sun, Li, & Zhang, 2011).
In addition, trehalose-6-phosphate synthase is involved in the complicated cold adaptation process in T. pui (Min et al., 2016). To obtain a further understanding of cold adaptation, two D9D genes (TpdesatA and TpdesatB) were sequenced in T. pui larvae and their expression patterns were investigated by real-time PCR during different seasons and cold exposure under 0°C in the laboratory. The results might serve to build a framework for comprehensively understanding the biology and molecular mechanisms of T. pui adaptation to thermal stress.

| Temperature measurement of soil with T. pui larvae
Temperature of soil at 20 cm below the surface was measured with Hobo Pro temperature and RH data logger (Model H08-032-08, Ecotech Co. LTD, USA). The data logger was set to record the tempera-

| Insect collection and cold exposure regime
The investigation of seasonal expression patterns was processed from July 2008 to June 2009. In the middle of every month, six individuals of the sixth instar T. pui larvae were collected from Mt.
Segrila (4,156 m, 29°37′N, 94°37′E) in the Tibetan Plateau, and these samples were used to transcription level analysis. Experiments under cold exposure were processed from July to August in 2013.
More than 100 individuals of sixth instar T. pui larvae were collected in July 2013 at the same area, then fed in soil at laboratory, and the environment-controlled at 10°C; after fifteen days, they were used to cold exposure experience. Ten individuals were used as a control group. The other were reared at 0°C (Thermo Scientific Precision, USA) and collected at different times, including short term (1, 3, 6, 12 hr), midterm (24, 48, 72 hr, 5 days), and long term (7, 10, and 15 days).

| Collection of fat body
All samples were dissected to obtain fat bodies. The fat bodies isolated from two larvae were mixed and then stored in RNA protect solution (TaKaRa, Japan) at −80°C.

| Cloning the full-length cDNA of two D9D genes
Total RNA was extracted from the fat body of one individual using

| Sequence analysis
The obtained fragments of two D9D genes were assembled by DNASTAR and the ORFs were identified through ORF Finder (Thompson, Higgins, & Gibson, 1994), respectively. Amino acid sequences were deduced from the corresponding cDNA sequences by using the translation tool on the ExPASy Proteomics Web site,  (Kayukawa et al., 2007), phylogenetic tree was constructed using the neighbor-joining method in MEGA software with 1,000 bootstrap replications.

| Quantitative analysis of two D9D genes
The seasonal expression and cold adaptation changes of two D9D genes were investigated through RT-PCR in CFX96™ Real-Time System. Two pairs of primers were designed for the quantitative analysis of two genes as well as a pair of primers for the control (β-actin) (

| Statistical analysis
Means and variances of treatments were analyzed using SPSS program (version 19.0, IBM Inc., USA), and the relative mRNA levels of D9D in July or control group was set as 1. All data were shown as mean ± SD. The means were compared with variance (ANOVA) and Tukey's studentized range test with the level of significant difference at p < .05 and highly significant difference at p < .01.

| Sequence identification and characterization of two D9D genes
The full length of two D9D genes, TpdesatA and TpdesatB, were ob-

| Seasonal expression patterns of two D9D genes
Quantitative analysis was performed to indicate the seasonal expression patterns of two D9D genes through RT-PCR. The soil temperature kept low level in whole year ( Figure 5a). As shown in

| Expression patterns of two D9D genes during cold exposure under 0°C
0°C was set to explore the cold adaptation mechanism under stable cold exposure in laboratory. Significant change was detected in the expression level of TpdesatA during the cold stress (F 11, 35 = 40.777, p < .001). In the short-term and midterm cold exposure, TpdesatA was stable, and remained at a low level from 1 hr to 5 days with no substantial change detected (Figure 6a). In the long-term cold exposure, the expression of TpdesatA increased from 5 days to the highest level at 10 days (2.43-fold) and slightly declined to 1.88-fold at 15 days. Expression of TpdesatB was significantly affected by cold exposure (F 11, 35 = 109.469, p < .001), the expression of TpdesatB was up-regulated from 6 hr (2.55-fold) to 5 days (2.97-fold) with a highest level appeared at 24 hr (3.59-fold), and TpdesatB was down-regulated before 3 hr and after 7 days.

| DISCUSSION
As a plateau insect with high cold tolerance, a series of physiological and biochemical mechanisms are evolved in T. pui. The proteins, total sugar, and total fat in the hemolymph as well as the fatty acid in whole body had the negative correlation with soil temperature . Moreover, trehalose-6phosphate synthase, HSP90 of T. pui, rather than HSP70, responds to temperature changes (Min et al., 2016;Zou et al., 2011). In this paper, TpdesatA and TpdesatB were found to have the relation with cold tolerance of T. pui.
F I G U R E 2 Nucleotide and deduced amino acid sequences of TpdesatA (a) and TpdesatB (b) The start and stop codons were showed as bold. Four transmembrane domains and three conserved histidine residues were boxed and underlined, separately. Five hydrophobic regions were leaned and underlined

F I G U R E 3 Multiple alignment of
Tpdesat amino acids in insects Four transmembrane domains and three conserved histidine residues were boxed and underlined, separately. Five hydrophobic regions were leaned and underlined In T. pui larvae, the lipid content changes in response to soil temperature. In phospholipids, C18:1 and C18:2, showed significant negative correlation with soil temperature. However, the fluctuation soil temperature did not cause any significant changes in any of the individual's triacylglycerols fatty acids , to which the molecular mechanism remains unknown. D9D plays an essential role in cold hardiness, by increasing the ratio of unsaturated and saturated fatty acids (UFA/SFA) (Rinehart et al., 2010;Tiku et al., 1996). In this study, two D9D genes were isolated for the first time in Thitarodes insects, and their expression patterns were investigated during different seasons and cold exposure under 0°C.
The two D9D genes separately encoded 346 AA and 359 AA amino acids, which correspond to the size range in other insects from NCBI. Alignment of two D9D genes and isoforms of thirteen other insects revealed D9D genes exist in several highly conserved regions.
Four transmembrane domains existed in D9D, suggesting that the sequence spans the lipid bilayer of membranes four times (Kayukawa et al., 2007;Los & Murata, 1998). Three histidine residues in these D9D genes were the highly conserved regions which are catalytically essential in desaturases (Shanklin, Whittle, & Fox, 1994). These histidine residues are suggested to combine with iron atoms at the catalytic center (Los & Murata, 1998). According to the N-J tree that was constructed in current study, two D9D genes in T. pui occurred in two independent clades, indicating that two D9D has different substrate specificities.
D9D desaturase is a key enzyme in synthetic pathway of UFAs, contributing to the formation of C16:1, C18:1, and C18:2. And these three UFAs are crucial to sustaining the fluidity of membranes under cold conditions (Kayukawa et al., 2007;Khani et al., 2007;Miyazaki, Kayukawa, Chen, Nomura, & Ishikawa, 2006). Previous investigations had proved that D9D gene was critical for cold adaptation in fish (Tiku et al., 1996), bacteria (Sakamoto & Bryant, 1997), and plants (Vega, Del Rio, Bamberg, & Palta, 2004). In this study, the expression level of TpdesatA was up-regulated in the long-term cold exposure and remained at a low level at short term and midterm; this indicates that TpdesatA contributes to long-term cold hardiness.
It was shown that seasonal expression patterns of TpdesatA exhibited a negative correlation with soil temperature (r = −.388, p = .390). Kayukawa et al. (2007) proved that the expression of D9D increased to enhance the cold hardiness in Delia antiqua through up-regulating the abundance of C16:1 and C18:1. So far, the same results were seen in S. crassipalpis (Rinehart et al., 2010), A. albopictus (Reynolds, Poelchau, Rahman, Armbruster, & Denlinger, 2012) and F. candida (Waagner et al., 2013). At the same time, seasonal cold-hardening is defined as cold-hardening that requires at least days to weeks for induction (Teets & Denlinger, 2013). Therefore, we suggest that TpdesatA has contributed to seasonal cold-hardening. In phospholipids of T. pui larvae, C18:1 was the most abundant UFAs and exhibited a weak negative correlation with soil temperature. C18:2, the second abundant UFA, was highly accumulated at early days of overwintering and fluctuated in lower levels during warmer seasons. Prewinter accumulation was also detected in C18:3 . These three UFAs content change in phospholipids may associate with the regulation of TpdesatA. Moreover, C16:0 was the second abundant in triacylglycerols, with a significant negative correlation with soil temperature . At the same time, TpdesatA gene was clustered in the △9 (16 = 18) group in phylogenetic tree. It indicates that TpdesatA works in seasonal cold-hardening, and C16:0 and C18:0 served as main substrate in triacylglycerols and phospholipids, respectively.
During cold exposure at 0°C, TpdesatB up-regulated from 6 hr to 5 days and down-regulate after 5 days; this indicates that TpdesatB may responds to cold temperature in short-term cold exposure. The same results were obtained in the winter diapause pupae of D. antiqua (Hao et al., 2012 (Joanisse & Storey, 1996;Teets & Denlinger, 2013). Therefore, TpdesatB may act as important short-term regulate substance in cold exposure and caused the change of the proportion of fatty acids in the body. Meanwhile, TpdesatB rather than TpdesatA was clustered closer to the △9 (18 > 16) group in phylogenetic tree. It indicated that C18 served as the substrate of TpdesatB prior to C16 in the short-term cold exposure.
Our results suggest that, during cold exposure at 0°C, TpdesatA and TpdesatB contributed to the cold tolerance in T. pui larvae.
TpdesatA has contributed to cold hardiness in long term and TpdesatB