Response Of Bird Cherry-oat Aphid,Rhopalosiphum Padi (L.) To Thermal Stress And Molecular Mechanisms

The bird cherry-oat aphid, Rhopalosiphum padi(L.)(Hemiptera: Aphididae), is a destructive wheat pest wordwide. This aphid not only damages by sucking plant directly and secreting honeydew causing sooty mould, but also by transmitting the Barley yellow dwarf virus(BYDV). The pest caused serious economic losses to wheat productions every year in the world. R. padi has adapted to a wide range of temperatures, and shows higher temperature resistance than other wheat aphids. It distributes in all the wheat planting areas in China, especially in the Yangtze River Basin and the Yellow River basin. However, it is damaging wheat more and more seriously in the northern China, which is considered a consequence of warmer winter that improve the possibility of overwinter of the pest in the north.Temperature adaptation of insect is an important branch of Entomology. Temperature adaptation is related to the response of insect to temperature stress, the thermotolerance and adaptive evolution to environment of insect species, which can affect the population dynamics, the geographic distribution, and the species displacement of insect. Research of temperature adaptation can provide the theoretical basis for forecasting insect pest outbreaking, analyzing geographic expansion of insect pest and development of reasonable control strategies under global warming. In the current study, we analyzed the effects of high temperature on population dynamics, basing on the effect of survival, fecundity and development of R. padi under high temperature stress and short-time high temperature exposure. Seven heat shock protein genes(Hsp) were successfully cloned from R. padi. Expression level of five R. padi Hsp genes were analyzed at different wing morphs, different developmental stages, different reproductive modes and after heat stress. Expression levels of five R. padi Hsp genes were analyzed after heat hardening and cold acclimation. The effects of heat hardening on thermotolerance, fecundity and expressions of Hsp genes were also investigated. Furthermore, we analyzed the expression levels of five R. padi Hsp genes when exposure to sublethal concentration of beta-cypermethrin. The main results are as follows:1. Effects of short-term high temperature on R. padi survival and reproductionThe survival rates of apterous and alate virginoparae adults decreased with the increase of temperature and time. Moreover, apterous virginoparae adult is more heat-tolerant than alate. After short-term high temperature exposure, the longevity of R. padi F0 generation was significantly shortened, whereas the fecundity was decreased significantly. However, longevity of R. padi F1 generation was not significantly different. The fecundity of R. padi F1 generation after treatment at 36 °C and 38 °C were significantly less than the control and the treatment at 37 °C. At the treatment of 38 °C, the development duration of nymph for F1 generation was remarkably prolonged, whilst the net reproduction rate(R0), the intrinsic rate of increase(rm) and the finite rate of increase(?) of F1 generationwere significantly lower than the control. The current results indicated that short-term high temperature exposure has significant negative effects on the R. padi F0 generation population, and the population growth of R. padi F1 generation was inhibited under short-term treatment of 38 °C.2. Cloning and sequences analyses of Hsp genes from R. padiSeven Hsp genes(RpHsp60, RpHsc70, RpHsp70-1, RpHsp70-2, RpHsc70-3, RpHsc70-5 and RpHsp90) were successfully cloned from R. padi. Amino acid sequence analysis showed that these Hsp genes had signature sequences of Hsp60 family, Hsp70 family and Hsp90 family, respectively. Meanwhile, RpHsp60 and RpHsc70-5 belong to the mitochondrial Hsp family, RpHsc70, RpHsp70-1, RpHsp70-2, and RpHsp90 belong to the cytoplasmic Hsp family, and RpHsc70-3 is an endoplasmic reticulum Hsp70 gene. Sequence similarity analysis showed that sequences of seven Hsp genes are highly conserved, and were highly homologous with those of the corresponding protein sequences of Hsp genes from other Aphididae aphids.3. Relative expression of Hsp genes at different wing morphs, different developmental satges and different reproductive modes of R. padiThe relative levels of five Hsps(RpHsp60, RpHsc70, RpHsp70-1, RpHsp70-2 and RpHsp90) significantly changed among different wing morphs, different developmental stages, different reproductive modes and different aphid forms. RpHsp70-2 expression level in alate virginoparae adult of anholocyclic type was significantly higher than that of apterous. There is no significant difference in expressions of five Hsp genes between the two wing morphs in holocyclic type. However, RpHsp70-1, RpHsp70-2 and RpHsp90 transcripts in holocyclic type were significantly higher than that of anholocyclic type. RpHsp60 was highly expressed at nymph stages. RpHsc70 expression levels were not significantly different among different aphid forms. The expressions of RpHsp70-1 and RpHsp70-2 in male were significantly higher than that of other aphid forms and control, while RpHsp90 transcript in eggs was the highest. Our results showed that the expressions of Hsp genes may play different role in the development and reproduction physiology of R. padi.4. Relative expression of R. padi Hsp genes under heat stressFive Hsp genes(RpHsp60, RpHsc70, RpHsp70-1, RpHsp70-2 and RpHsp90) were up-regulated under heat stress. After heat shock for 1 h, the expression of these five genes peaked at 36 °C in alate virginoparae adults, whereas peaked at 37 °C or 38 °C in apterous. Moreover, the expressions of Hsp genes decreased by 39 °C and 40 °C exposure, indicating that the response of Hsp genes to heat is limited. The expression patterns of five Hsp genes were varied after exposure to 36 °C for 1-5 h, but the expression levels were relatively stable. The response of five Hsp genes to heat stress were different, namely RpHsp70-1 and RpHsp70-1 were most sensitive to heat stress, RpHsp90 second, RpHsp60 and RpHsc70 were less sensitive to heat stress.5. Effects of temperature pretreatment on the expression levels of R. padi Hsp genesHeat hardening remarkably improved survival rates and the expressions of Hsp genes(RpHsp60, RpHsc70, RpHsp70-1, RpHsp70-2 and RpHsp90) at subsequent lethal temperatures, while the expression patterns of Hsp geneswere different after cold acclimation. The expressions of RpHsp after heat shock were up-regulation but were not significantly different to the control, whilst the five Hsp genes transcriptions were significantly induced by heat hardening and heat shock after heat hardening. RpHsp60 and RpHsc70 expressions in both wing morphs showed no significant difference compared with control after cold acclimation, indicating the two genes were insensitive to cold acclimation. The expression of RpHsp90 was remarkably induced by cold acclimation. Nevertheless, RpHsp70-1 and RpHsp70-2 expression in apterous virginoparae adults were significantly decreased. The results showed that the five Hsp genes may play different roles in low temperature adaptation.6. The trade-off between thermotolerance and fecundity of R. padiThe thermaltolerance, the fecundity and the expressions of five Hsp genes of R. padi were analyzed after temperature treatments. Results showed that Hsp genes play an important role in developing the temperature tolerance of R. padi. Mild hardening significantly increased heat tolerance, but didnot increase cold tolerance. However, the fecundity of the first ten days was significantly decreased, indicating improvement of thermotolerance was at the cost of low fecundity. Overexpression of RpHsp70-1, RpHsp70-2 and RpHsp90 increased the thermotolerance at the cost of decreasing in fecundity.7. Relative expression of R. padi Hsp genes when exposure to sublethal concentrations of beta-cypermethrinThe expressions of five Hsp genes(RpHsp60, RpHsc70, RpHsp70-1, RpHsp70-2 and RpHsp90) showed different profiles after exposure to sublethal concentrations LC10(0.3987 mg/L) and LC30(0.9280 mg/L) of beta-cypermethrin for 12 h, 24 h and 36 h. RpHsp60 expression was significantly down-regulated after treatment and had no significant difference among post-exposure for 12 h, 24 h and 36 h, indicating that RpHsp60 cannot be induced by beta-cypermethrin. Under exposure to two sublethal concentrations(LC10 and LC30) of beta-cypermethrin, RpHsc70, Rp Hsp70-1, RpHsp70-2 and RpHsp90 expressions were induced and reached a maximum at 24 h after exposure. Moreover, expression levels of Hsp genes under LC10 and LC30 of beta-cypermethrin were significantly different, which indicated that respose of Hsp genes to beta-cypermethrin was time- and concertration-dependent. In addition, the responses of RpHsc70, RpHsp70-1, RpHsp70-2 and RpHsp90 were more sensitive to heat stress than beta-cypermethrin, suggesting possible different response mechanisms of RpHsp involved in thermal stress and beta-cypermethrin exposure.