56 ISJ 14: 56-62, 2017 ISSN 1824-307X RESEARCH REPORT Gene expression of HSP90 and HSP70 in four silkworm hybrids (Bombyx mori L.) in response to severe thermal shock SF Mousavi1, SH Hosseini Moghaddam2, N Ghavi Hossein-Zadeh1, SZ Mirhosseini2 1Department of Animal Science, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran 2Department of Animal Science and Department of Sericulture, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran Accepted March 6, 2017 Abstract Usually silkworm egg producers provide several silkworm hybrids with different qualities of productivity and viability traits. This study was conducted to compare the expression of HSP70 and HSP90 genes among different silkworm hybrids. In the fourth day of fifth larval instar of two Japanese maternal parents (103×104 and 107×110) and two Chinese maternal parents (110×107 and 104×103), silkworm larval fat body was sampled from heat shock (45 °C for 35 min) and non-heat shock groups. Sampling was done at 0, 2, 4 and 24 h after heat shock. Gene expression of HSP70 and HSP90 (target genes) were measured by qRT-PCR using RPL27a as reference gene. The prolonged heat shock (8 h at 39 °C) was utilized to examine the thermal tolerance of larvae in comparison with control group (24°C). The results showed both HSP70 and HSP90 have been up-regulated in the treated larvae. The hybrids with Japanese female parents (107×110 and 103×104) were more sensitive than two others; moreover in these hybrids, HSP90 and HSP70 were expressed significantly more than others after heat exposure. The maximum expression was occurred in the time zero, then a decreasing trend was observed over time. Comparison of larval mortality among four hybrids revealed that (103×104) and (110×107) hybrids had the highest and the lowest mortality rate. Key Words: gene expression; heat shock proteins; real-time PCR; silkworm hybrids; thermotolerance Introduction Sericulture has an important role in village economy in countries like Iran where sustainable rural development is very important. High sensitivity of commercial hybrids to high temperature is the main barrier in its expansion in hot and dry climates. Therefore, factors affecting silkworm tolerance should be considered in the silkworm breeding and introduction of new hybrids (Hosseini Moghaddam, 2005). Moreover, substantial studies have been carried out by researcher to evaluate the effect of heat stress on various organisms due to probability of sudden and rapid climate change when the earth is getting warmer. When insects expose to extreme temperature, they respond to this temperature change by arising heat shock proteins (HSPs) as molecular chaperones to protect of protein folding process. HSPs family is divided according to their molecular __________________________________________ Corresponding author: Seyed Hossein Hosseini Moghaddam Department of Sericulture, Faculty of Agriculture Sciences University of Guilan, Rasht, Iran E-mail: hosseini@guilan.ac.ir weight, function and sequence homology into several groups including HSP100, HSP90, HSP70, HSP60, HSP40, small HSP (sHSPs) and HSP10(Zhao and Jones, 2012). Among these HSPs, high molecular weight HSP70s as a housekeeping molecule has different responsibilities in insects such as developmental processes and fecundity (Jensen et al., 2014), development (Huang et al., 2009), diapause (Rinehart et al., 2007), longevity (Choi et al., 2014), and metamorphosis (Zheng et al., 2010). HSP70s genes were up-regulated in response to cold or heat stress in a variety of insects, including Coleoptera (Mahroof et al., 2005), Diptera (Gray, 2013) and Lepidoptera (Jiang et al., 2012; Choi et al., 2014; Shen et al., 2014). In a study on flash fly (Sarcophaga crassipalpis) in response to hypoxia, HSP90 showed little response, however, HSP70 was the most responsive and increased several hundred fold in the cells (Michaud et al., 2011). Results of previous studies on the Chilo suppressalis, Liriomyza trifolii, and Pteromalus puparum showed that HSP70 can be related to thermotolerance and survivability (Cui et al., 2010; Zheng et al., 2010). In Drosophila melanogaster, mailto:hosseini@guilan.ac.ir 57 family of HSP70’s could not make thermotolerance against severe thermal shock (Bettencourt et al., 2008). The well-defined role of HSPs in acquired thermotolerance in the silkworm and other insects is not clearly known yet (Manjunatha et al., 2010). HSP90 that forms about 1 - 2 % of cellular proteins is one of the most abundant proteins in the living cells. When insects are under normal conditions, the HSP90 expressed at low levels (Wegele et al., 2004). HSP90 acted as a protective against thermal stress in some insects including Plutella xylostella and Liriomyza huidobrensis, (Sonoda et al., 2006; Huang and Kang, 2007). Zhang et al. (2009) reported that among different tissues of silkworm, sometimes the HSP90 gene expression was up-regulated and sometimes was down-regulated. The commercial silkworms are usually two- way cross hybrids (direct and reciprocal crosses) which are synthetized by two parents including Japanese (e.g. 103, 107) and Chinese (e.g. 104, 110) strains. According to previous report (Hosseini Moghaddam, 2005) the productivity traits (cocoon weight and cocoon shell weight) of Japanese maternal parents (103×104 and 107×110) were higher than Chinese maternal parents (110×107 and 104×103) and conversely for viability traits. The purpose of this research is the comparison of gene expression of two high molecular weight HSPs in four commercial silkworm hybrids (including two maternal Chinese and two maternal Japanese parents), besides the relationship of gene expression with thermal tolerance of silkworm larvae. Materials and Methods Silkworm rearing, thermal treatment and sampling Two Iranian silkworm hybrids: 110×107 and 104×103 (first parent is female and a Chinese cocoon shape strain) and their reciprocal 103×104 and 107×110 (first parent is female and a Japanese cocoon shape strain) were reared in the Iran Silk Research Center. The 103 and 104 silkworm strains are originally from Japan and the strains 107 and 110 were isolated from a Korean hybrid under FAO/UNDP TCP project (1992-1997). Fifth instar larvae were transferred to the silkworm Laboratory, Faculty of Agricultural Sciences, University of Guilan to continue rearing and heat shock treatments. Sampling was done from fat body. This tissue is under larval skin that can easily affected by heat exposure. Fat body as a site for energy storage tissue in the silkworm, synthetize many biological proteins like heat shock proteins that these substances are expected to be active in the fat body (Kajiwara et al., 2006). While all process was performed on ice, the fat body free of muscle was collected in the fourth day of fifth larval instar from both heat shock (45 °C for 35 min) and non-heat shock larvae at 0, 2, 4 and 24 h after heat treatment. Before collection of fat body, the surface of dissected larva was washed by ice- cold insect physiological salt solution (0.7 % NaCl) (Chavadi et al., 2006). Three independent fat body samples were mixed together to minimize variation and to get enough amount of samples. They were store immediately at -80 °C for further use. RNA Extraction, cDNA synthesis and Real time-PCR RNA extraction procedure was carried out by TRIzol reagent (Invitrogen, USA) based on the supplier’s instructions. The quality and quantity of extracted RNA was evaluted using NanoDrop-2000 spectrophotometer (Thermo Scientific) and the quality of agarose gel electrophoresis bands. DNAse I treatment (TAKARA, Japan) was applied to avoid possible contamination of genomic DNA. The cDNA of all samples were made using cDNA synthesis kit (Thermo Scientific) based on manufacturer protocol. Specific primers of HSP70 and HSP90 were designed using online software Primer 3 (Table 1). Real time-PCR was carried out using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific). Data was normalized using Livak (2001) formula (2-ΔΔCt). A factorial experiment (4×4) with completely randomized design was utilized to evaluate the effects of different hybrids (110×107, 104×103, 103×104, 107×110) and the times after heat shock (0, 2, 4, 24 h), as fixed effects, and their interactions on gene expression data. Statistical analysis of data was performed using the GLM procedure of SAS 9.0. Least squares means were used to identify the main effects. Table 1 Specific primer sequences for Real-time PCR Gene NCBI Reference Primer Sequence PCR Product length Annealing temperature RPL27a NM_001044057 Right : TGACAGGTTGTTTGGGGAG Left: CAGACGAGGCTGAAGTATGC 144 60 HSP70 NM_001043931.1 Right: GTGCTTCATGTCCTGCTGAA Left: TCGCCTTGAACCCTAACAAC 100 60 HSP90 NM_001043411.1 Right: AGGCCTTCGAACTTCACCTT Left: ATGGCAAGACCCTTGTATCG 103 54.7 58 Table 2 Least-squares means of HSP70 and HSP90 gene expression in the studied silkworm hybrids at four times after thermal exposure Expression of HSP90 Expression of HSP70 Times Hybrids 2183.49a 1549.54a 0 107×110 595.96b 646.83b 2 107×110 83.54c 1.002e 4 107×110 1.85c 0.01e 24 107×110 32.78c 277.47c 0 110×107 7.26c 7.51e 2 110×107 0.13c 0.06e 4 110×107 0.04c 0.007e 24 110×107 278.11a 1598.42a 0 103×104 49.90c 26.07 d 2 103×104 2.15c 21.11 d 4 103×104 0.43c 0.09 e 24 103×104 26.99c 97.57 d 0 104×103 20.87c 85.62 d 2 104×103 10.06c 2.28 e 4 104×103 0.01c 80.86* 0.07 e 27.78* 24 104×103 SE a-e Different superscripts within a column indicate significant differences (p < 0.0001) * Standard error for all least squares means of gene expression Evaluation of silkworm viability and thermal tolerance In order to measure thermotolerance of different silkworm hybrids, larvae were exposed to 39 °C for 8 h (9 am to 5 pm) from second to seventh day of fifth instar as long-term heat stress; in other word six days from one day after forth molting to one day before larval cocooning. While larval mortality was recorded daily, mortality rate was measured after two days (LO1) and also at the end of heat shock period (LO2). Because, it is expected that sensitive genotypes will respond more quickly to the initial thermal shock and die; therefore, the first time measurement of mortality rate (LO1) was considered as a thermotolerance criterion. In the same time, larval mortality was recorded for non- heat stress groups. LO2 is not a good criterion to rank the silkworm hybrids; since long-term heat exposure may cause to be killed most of larvae in each tray. Average larval mortality in response to heat shock was reported as log-transformed values to assure normality. A factorial experiment (4×2) with completely randomized design was utilized to evaluate the effects of different silkworm hybrids (110×107, 104×103, 103×104, 107×110) and heat exposure (with or without thermal shock), as fixed effects, and their interactions on larval mortality (LO1 and LO2). Statistical analysis of data was carried out using the GLM procedure of SAS 9.0. Least squares means were used for identifying the main effects. Results The results showed that gene expression of HSP90 and HSP70 in hybrids with Japanese maternal parents) (103×104 and 107×110) were significantly (p < 0.0001) higher than hybrids with Chinese maternal parents (104×103 and 110×107). Table 2 shows that the fluctuation of HSP expression for both genes is almost the same. In other word, the expression of HSP90 was in good accordance with HSP70. Least-squares means of HSP70 and HSP90 gene expression (Table 2) indicated that there was a high significant difference(p < 0.0001) in the HSP70 and HSP90 expression between two reciprocal hybrids (110×107 vs. 107×110 and 103×104 vs. 104×103) immediately after heat exposure (Fig. 1). Expression of both genes immediately after heat shock (time zero) was significantly higher than other times (p < 0.0001) in all genotypes, afterwards, gradually decreased to the lowest level after 24 h (Table 2). In this time down-regulation was observed in almost all genotypes, in fact, HSP70 and HSP90 expression reduced over time. Four h after thermal exposure, while HSP70 expression in the three hybrids declined but still 103×104 had higher expression. The hybrid 104×103 had significantly lower expression among hybrids (p < 0.0001) which proposed that this hybrid need less HSP70 and HSP90 in thermal shock conditions. In another experiment a prolonged heat shock (8 h at 39 ◦C for six days) was implemented to examine the thermotolerance of larvae. The mortality rate (in %) was considered as a measure of thermotolerance. Mortality of all treatment groups was higher than control groups. The hybrid 110×107 and 103×104 had the lowest and the highest mortality rates, respectively. In fact, 103×104 as a sensitive hybrid had the highest losses and 110×107 hybrid as a resistant hybrid had the lowest 59 Fig. 1 HSP70 and HSP90 gene expression immediately after heat exposure (time zero) in the studied silkworm hybrids losses (Fig. 2). The same result was obtained control group; however differences was not significant (Table 3). 110×107 and 107×110, two silkworm hybrids that their parents were originally from Korea, were more resistant than hybrids 103×104 and 104×103 with Japanese parents. Among four genotypes, the sensitive hybrids (103×104 and 107×110) had the highest gene expression immediately after heat exposure (zero time) implying that these genotypes need more HSP70 and HSP90 under severe thermal conditions. In fact, hybrids with Japanese maternal parents (107×110 and 103×104) were sensitive and hybrids with Chinese maternal parents (110×107 and 104×103) were relatively resistant. Therefore, sensitive hybrids immediately after heat exposure need both HSP70 and HSP90 proteins to reduce thermal effects. Discussion A large difference was observed between the genotypes in terms of HSP expression. Generally, the results showed that the hybrids with Japanese maternal parents had more HSPs expression than Chinese maternal parents. There are some reports on genetic differences of HSP70 or HSP90 among silkworm genotypes such as comparison of Nistari and Jingsong (Hosseini moghaddam et al., 2008), Nistari and NB4D2 (Velu et al., 2008), C. Nichi, Pure Mysore and NB4D2 strains (Joy and Gopinathan, 1995) and Pure Mysore and NB4D2 (Sosalegowda et al., 2010). Velu et al. (2008) reported little differences in HSP70 expression between two resistant and sensitive strains on agarose gel (semi- quantitative) after a mild heat shock (41 °C for one hour) but Li et al.)2011(using qRT-PCR method showed that expression of HSP70 in Jingsong (a thermosensitive commercial strains) was significantly more than Nistari breed )a thermotolerant multivoltine breed in tropical region). It means that thermo-sensitive breed induced strongly HSP70 mRNA after heat shock treatments. This result was consistent to our study which among four genotypes, the sensitive hybrids (103×104 and 107×110), as Japanese maternal parents, had the highest gene expression. Garbuz et al. (2002) reported that among Drosophila species and strains originating from different climatic zones, some of species and strains exhibited positive correlation between HSP70 expression and thermotolerance and for others negative correlation. Table 3 Average larval mortality in the four silkworm hybrids for both thermal treatment and control groups Hybrids LO 1* LO 2 107 ×110 (Treatment) 0.70 ± 0.07c 1.84 ± 0.02a 107×110 (Control) 0.13 ± 0.02d 0.93 ± 0.01d 110 ×107 (Treatment) 0.50± 0.08cd 1.66 ± 0.01b 110×107 (Control) 0.09d 0.57 ± 0.005e 103×104 (Treatment) 1.66± 0.07a 2.02± 0.003a 103×104 (Control) 0.34 ± 0.02d 1.23 ± 0.07c 104 ×103 (Treatment) 1.19± 0.03b 1.91± 0.005a 104 ×103 (Control) 0.09d 0.7 ± 0.04d a-e Different superscripts within a column indicate significant differences (p < 0.0001) * Average larval mortality is log-transformed values (LO1: First period of measuring mortality; LO2: Second period of measuring mortality) 60 Fig. 2 Larval mortality percentage by treatment and control groups in the studied silkworm hybrids (LO1: First period of measuring mortality; LO2: Second period of measuring mortality; Average larval mortality is log-transformed values) In the current study HSP70 and HSP90 expression was up-regulated in the fat body of silkworm larvae after severe (45 °C) but non-lethal heat shock. . Li et al. (2011) observed higher expression of HSP70 in fat body rather than testis and ovary in a thermosensitive strain of silkworm. Velu et al. (2008) reported mid gut and fat body tissues had higher HSP70 expression than the cuticle and silk gland tissues. Keshan et al. (2014) studied HSP90 expression in various tissues of silkworm and indicated that a higher HSP90 expression in all examined larval tissues after mild (39 °C) and mild to severe (42 °C) heat treatments. The highest expression was observed just immediately after heat exposure (time zero) in all silkworm hybrids. To our knowledge, this is the first study where the differences of HSP gene expression were evaluated for reciprocal crosses in the silkworm. In the silkworm, high thermotolerance in fifth instar larvae reflects its adaptation to high temperature (Manjunatha et al., 2010). A large difference was also observed between the genotypes in terms of heat tolerance. The hybrids with Japanese maternal parents were more sensitive than the Chinese ones, which was in line with other researches (Vasudha et al., 2006; Firdose and Reddy, 2009). Hsieh et al. (1995) by comparing Chinese and Japanese maternal parents demonstrated that Feng, a Chinese strain, was the most tolerant strain followed by Japanese races, Kuo and J-09, while another Chinese strain, C-54 was most susceptible. In different studies, different genotypes had different reactions to the heat stress. This diversity among silkworm varieties is related to their genetic background and related silkworm breeding plan. Our results showed that this kind of differences could be detected in molecular levels. Further investigations of these differences can help us to understand the mechanisms of protecting cells against environmental stresses and also identify variation of HSPs genes expression among susceptible/ tolerant strains and hybrids of silkworm. Conclusion The results of this study showed that both HSP70 and HSP90 have been up-regulated in the treated larvae. The hybrids with Japanese female parents (107×110 and 103×104) were more thermo- sensitive than two others, moreover HSP90 and HSP70 were also significantly expressed in these two hybrids after heat shock. Comparison of larval mortality among four hybrids revealed that the hybrid 103×104 had the highest mortality rate and 110×107 had the lowest mortality rate. High expression of HSPs in thermo-sensitive hybrids showed importance of HSPs in survivability. Generally, it can be concluded that the expression patterns of high molecular weight HSP can 61 determine the various levels of thermotolerance in the different silkworm genotypes. Acknowledgement We are grateful to the Iran Silk Research Center (Guilan province) for cooperation in silkworm rearing. This work was supported by biotechnology committee of University of Guilan under their block grant. 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