Response To Cold Stress In The Pink Borer, Sesamia Inferens (Walker) (Lepidoptera:Noctuidae)

The pink borer, Sesamia inferens (Walker) (Lepidoptera:Noctuidae), is a major rice pest in China, Korea, Japan, India, Philippines, Vietnam, Laos, Cambodia and South Pacific islands (Papua New Guinea, etc.). According to the past researches, the north limit of S. inferens distribution is roughly in the north latitude 34° in our country. With global warming, S. inferens was further dispersed to northern district. S. inferens harm on rice has occurred in the north of latitude 34°. Before the latter part of the last century, S. inferens was a minor pest on rice. In recent decades, S. inferens became one of major pests of rice with its damages increasing.This raises a series of questions. How physiological responses of S. inferens reacted in overwintering process? Which cold tolerant strategy of S. inferens overwintering in the field? What kind of role did S. inferens heat shock proteins (HSPs) playing in in cold tolerant? Unfortunately, overwintering S. inferens researches were only reflects the characteristics of a specific period of overwintering process. The body of research on the cold tolerance of S. inferens lacks comprehensiveness. In addition, from molecular biology level, especially in HSPs research of S. inferens have never been studied.Therefore, in this study, the cold tolerant characteristics and strategy of S. inferens overwintering in the field was studied from the Angle of ecology and physiology. And the role of 5. inferens HSPs in cold tolerant was also studied. Main conclusions are as follows:1 Cold tolerance characteristics and strategy of overwintering Sesamia inferens in YangzhouThe relation between temperature and mortality was fitted to a curve: M=0.9832e-0.0354(T+12)-0.0004(T+12)4T= [-12,-2]. Beyond this range, accuracy decreased relationship. The LTemp50 and LTemp90 values were calculated as-6.119℃ and-9.774℃.We found that the cold tolerance of overwintering larvae varied significantly. Maximum S. inferens cold tolerance was observed in larvae collected on January 30,2013. However, the SCPs (supercooling points) of larvae varied insignificantly, with an average value of-6.80℃. Before March 9,2013, larvae water content stabilized at a low level, with an average value of 63.5%, but it subsequently rose significantly, to 75.2%. Low molecular weight molecular sugars and polyols, closely related to such a freeze tolerant strategy, were increased from low levels to their peaks in January (glycerol,359.8μg/g; trehalose,20.5 mg/g; fructose,69.8μg/g; glucose,377.3 μg/g; myo-inositol,59.6μg/g), after which levels declined.As this study demonstrates, the Yangzhou population of S. inferens is a freeze-tolerant insect.2 Cloning on HSPs gene of Sesamia inferensFive heat shock protein genes were cloned from S. inferens by RT-PCR and RACE, and were named Sihsp83, Sihsc70, Sihsp21.4, Sihsp20.6 and Sihspl9.6, respectively. The complete cDNA of 5 HSP genes are 2496 bp,2160 bp,1385 bp,835 bp and 798 bp respectively, and their respective opening reading frames are 2154,1962,564,552 and 525 bp, which encode 717,653, 187,183 and 174 amino acids with predicted molecular weights of 82.6,71.6,19.6,20.6 and 19.6 kDa, respectively. Sequence analysis suggested that the five HSPs shared high similarity with those of other species and contained conserved motifs found in their respective family of other species. Conserved sequences of HSP90, HSP70 and sHSPs family were found in the amino acid sequence. The cytoplasmic localization signal in C-terminal was explained that S. inferens HSP83 and HSC70 were cytoplasmic heat shock proteins.Analysis of genomic DNA demonstrated that the Sihsc70 and Sihsp21.4 contained 2 and 1 introns, respectively. In Sihsc70, fist intron,685 bp, located upstream of the coding region, and no gap between the two, and second intron,803 bp, located in the coding region. In Sihsc21.4, the intron with 1210 bp, located in the coding region. But no introns were found in Sihsp83, Sihsp20.6 and Sihsp19.6.Finally, phylogenetic analysis suggested that the five HSPs were homologues of their respective family.3 Screening study of Sesamia inferens reference genesSeven housekeeping genes (partial sequence),18S ribosomal RNA (18S rRNA), elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S13 (RPS13), ribosomal protein S20 (RPS20), tubulin (TUB) and β-actin (ACTB), were cloned from S. inferens by RT-PCR. And these genes were evaluated for their suitability as normalization genes under different experimental conditions by qRT-PCR. The results indicated that the best sets of candidates as reference genes were as follows:three genes (RPS13, RPS20 and EFT) for different tissues (heads, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes and salivary glands); three genes (18S rRNA, EF1 and GAPDH) for developmental stages and sex (egg masses, the first, second, third, fourth, fifth and sixth instar larvae, male pupae, female pupae, one-day-old male adults and one-day-old female adults); three genes (18S rRNA, RPS20 and TUB) for fifth instars treated with different temperatures (27,-8,-6,-4,-2 and 0℃).4 Expression pattern analysis on HSPs gene of Sesamia inferensExpression patterns of five heat shock protein genes (Sihsp83, Sihsc70, Sihsp21.4, Sihsp20.6 and Sihsp19.6) in response to different tissues (heads, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes and salivary glands), developmental stage and sex (egg masses, the first, second, third, fourth, fifth and sixth instar larvae, male pupae, female pupae, one-day-old male adults and one-day-old female adults) and different temperatures (-8,-6,-4,-2,0 and 27℃) were examined by Real-time PCR. Results showed that five hsps were expressed under all experimental conditions. In different tissues, Sihsp83 and Sihsp19.6 were highest expressed in hindgut; Sihsp20.6 was highest expressed in foregut; Sihsp21.4 was highest expressed in heads; but expression of Sihsc70 varied insignificantly. In different developmental stage and sex,4 HSP genes, except Sihsp21.4, were highest expressed in eggs; Sihsp21.4 was highest expressed in female adults.5 HSP genes were induced up-regulation by different temperatures. Heat shock proteins were involved in the growth and development of S. inferens. Increased expression of heat shock protein genes were related to the increasing of S. inferens cold tolerance.