| The beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), is an important polyphagous insect. Originating from South Asia, it has spread to Europe, Africa, Australia, America and other parts of Asia. In South China including Guangdong, Shenzhen, Fujian province and Taiwan, the species exhibits stronger adaptability to high temperature and occurs continuously in full year without overwintering. Also, the infesting population usually peaks when local temperature is the highest. In addition to stronger heat tolerance, S. exigua also is sub-stronger cold-resistant by overwintering in temperate zones without entering diapause. The pupae have an average supercooling point of approximately -17℃with the lowest of -21℃in individuals. In China, the infesting population has spread northward to Liaoning province (123.29oE, 41.80oN) in Northeast China. Life history strategies including overwintering, migration and reproduction in S. exigua under the pressure of temperature, humidity, density and food were well documented by our previous study, showing a significantly environmental tolerance and ecological plasticity in this species. Furthermore, S. exigua is also well known as its rapidly increased resistance against many chemical pesticide and transgenic crops. Therefore, it could be presumed that S. exigua has stronger potential to acquire or evade tolerance to various environmental stresses. However, the biochemical and molecular mechanisms of this tolerance remain unknown.The heat shock proteins (HSPs), which were found in most organisms are highly conserved in all eukaryotes and prokaryotes so far studied. Up-regulation of Hsp70 and Hsp90 mRNA levels in response to cold and heat hardening, density, starvation, diapause and developmental stages were reported in several insects. However, there is no published information about the stress-induced HSPs gene sequences and their expression from S. exigua, which in turn, limits well understanding the mechanism of its ecological adaptability. This study is to examine the thermal responses among different developmental larvae and pupae of the species on the basis of cloning, sequencing and expression of Hsp90 and Hsp70. The results are as follows:Cloning of HSP70 and HSP90 full-length cDNA: According to the Hsp70 cDNAs of cabbage armyworm Mamestra brassicae ( GenBank: AB251894 ) , tobacco hornworm Manduca sexta(GenBank:AY220911), degenerate primers were designed. A full-length cDNA encoding Hsp70 from S. exigua was cloned and characterized by RT-PCR and RACE technique. The complete cDNA s (2315bp) contains a 2004bp open reading frame encoding 667 amino acid residues (GenBank accession no. FJ862049) and with a predicted molecular weight of 72.5 kD and an isoelectric point of 5.52. The nucleotide sequence of the cDNA is highly similar with the Hsp70 cDNAs of some other insect species, and it includes an important and intact HSP70 signature sequence.According to the Hsp90 cDNAs of cabbage armyworm Mamestra brassicae (GenBank: AB251894), armyworm Mythimna separate(GenBank: EU306519)and meadow moth Loxostege sticticali(sGenbank: EU233821), degenerate primers were design. A full-length cDNA encoding Hsp90 from S. exigua was cloned and characterized by RT-PCR and RACE technique. The complete cDNAs (2 453 bp) contains a 2154 bp open reading frame encoding 717 amino acid residues (GenBank accession no. FJ862050) and with a predicted molecular weight of 82.6 kD and an isoelectric point of 5.0. The nucleotide sequence of the cDNA is highly similar with the Hsp70 cDNAs of some other insect species, and it includes an important and intact HSP70 signature sequence.Cloning of Hsc70 cDNA partial sequence:According the Hsp70 cDNA of cabbage armyworm Mamestra brassicae (GenBank:AB251894), tobacco hornworm Manduca sexta(GenBank:AY220911),degenerate primers were designed. A partial-length cDNA encoding Hsc70 from S. exigua was cloned and characterized by RT-PCR The cDNAs (1541bp) encod 513 amino acid residues (GenBank accession no. GQ220728). The nucleotide sequence of the cDNA is highly similar with the Hsc70 cDNAs of some other insect species.The expression levels of Hsp70 and Hsp90 mRNA in larvae stressed by high temperatures: The plasmid with Hsp70 cDNA of S. exigua was used as a template. A real-time fluorescence quantitative RT-PCR was constructed to examine the transcriptional expression levels of Hsp70 from larvae (2, 3, 4, 5instar) of S. exigua under different high temperature stress (37, 39, 41, 43, 45℃) for 1 h, respectively,and corrected the result withβ-actin gene of S. exigua. The result showed that high temperature could induce Hsp70 gene expression in S. exigua larvae. Exposure the larvae under 43 and 45℃, the expression levels of Hsp70 gene can increase approximately 1000 times, and significantly higher than those of control(P<0.05). Mild temperature also can induce the expression of Hsp90 obviously, and the Hsp90 mRNA levels increase with increasing temperature in certain range. A significant increase in Hsp90 gene mRNA levels was detected in the fourth instar larvae exposed to 43 and 45°C(P<0.05).These results suggested that Hsp70 and Hsp90 play an important role in adaptation to cold temperature stress which can increase their tolerance to extracellular heat shock.The expression levels of HSP70 and HSP90 mRNA in larvae stressed by low temperatures: Hsp70 and Hsp90 mRNA levels were detected in larvae (2, 3, 4instar) exposed to 5,0,-10 and -15℃, respectively. The results showed that, Under the 0 and 5℃hardening for 3h, the expression levels of Hsp70 and Hsp90 were not significantly induced (P<0.05). However, when the larvae were stressed by -10℃for 2 h, the expression levels of Hsp70 and Hsp90 mRNA were significantly induced(P<0.05). These results suggested that Hsp70 and Hsp90 might be responsible for cold stresses of different intensity which can protect the cell. However the protective effect did not work exceed the the limit scope.The expression levels of Hsp70 and Hsp90 mRNA in pupae stressed bylow temperatures: Hsp70 and Hsp90 mRNA levels were detected using Real-time PCR in pupae (2, 3, 4-day pupa) exposed to 5, 0, -10 and -15℃, respectively. Although the pupae were exposed to 5, 0℃f or 3 h respectively, the Hsp70 and Hsp90 genes expression were not be induced significantly, they were significantly higher than those of control when pupae were hardened by -10℃for 2 h,. When the 2-day pupa were shocked at the critical temperature of -15℃for 2 h,, Hsp70 and Hsp90 gene expression levels were significantly higher than those of controls(P<0.05). These result showed that Hsp70 and Hsp90 may contribute to the cold hardening response.
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