| Xinjiang is currently the main cotton producing area in China,and its cotton planting area exceeds one-third of the country’s total cotton area.Hippodamia variegate(Goeze)(Coleoptera:Coccinellidae)and Propylaea quatuordecimpunctata(L.)(Coleoptera:Coccinellidae),the dominant natural enemy populations in cotton fields in Xinjiang,have good control effects on pests,such as Aphis gossypii.The preliminary field investigations found that there were only H.variegate in southern Xinjiang where the temperature was higher,while both H.variegate and P.quatuordecimpunctata were dominant populations in northern Xinjiang where the temperature was relatively low-temperature.Therefore,it is speculated that there are differences in the response and adaptation of the two ladybugs to high temperature,which in turn lead to differences in their geographical distribution.In this paper,H.variegate and P.quatuordecimpunctata were treated under the temperature of 32℃,35℃ and 38℃ to evaluate the effects of different temperature conditions on the survival,longevity,fecundity and predation ability of H.variegate and P.quatuordecimpunctata adults,so as to clarify the differences in their adaptability to high temperature.The physiological and biochemical responses of H.variegate and P.quatuordecimpunctata under different temperatures were compared to explore the difference of physiological response mechanism under high temperature stress.Then,the molecular regulation mechanism of the two ladybugs to heat stress was deeply analyzed by transcriptome sequencing and differentially expressed genes(DEGs)function analysis.Finally,RNA interference(RNAi)technology was used to identify the role of key genes in the process of ladybugs resistance to high temperature stress.The main findings are as follows:1.Effects of different temperatures on the adaptation of adults of H.variegate and P.quatuordecimpunctata.First,the differences in survival,longevity and fecundity of H.variegate and P.quatuordecimpunctata adults were analyzed at different experimental temperatures(32℃,35℃ and 38℃).The results showed that the adults H.variegate survival rate,longevity and fecundity were significantly higher than P.quatuordecimpunctata at all experimental temperatures.As the temperature increase,the survival rate,longevity and fecundity of P.quatuordecimpunctata decreased faster than that of H.variegate.The predation results showed that H.variegata consumed more cotton aphids than P.quatuordecimpunctata under all temperature treatments.With the increase of temperature,the predation ability of H.variegate showed a trend of "first increased and then decreased",and the predation ability was the strongest at 35℃(a:1.12),while the predation ability of P.quatuordecimpunctata continued to decrease(a:32℃:0.84;35℃:0.74;38℃:0.54).In conclusion,high temperature(35℃ and 38℃)had certain negative effects on survival,longevity,fecundity and predation ability of the two ladybugs,but the negative effects were more obvious for P.quatuordecimpunctata.2.Effects of different temperatures on physiological and biochemical reactions of adults of H.variegate and P.quatuordecimpunctata.The activities of four kinds of antioxidant enzymes(Superoxide dismutase,SOD;Peroxidase,POD;Catalase,CAT and Glutathione S-transferases,GSTs),total antioxidant capacity(T-AOC)and protein content of H.variegate and P.quatuordecimpunctata adults were analyzed at different temperatures(32℃,35℃ and 38℃).The results showed that with the increase of temperature,the activities of SOD and CAT increased,while the activities of GSTs decreased.The activity of POD in H.variegate first increased and then decreased,and the activities was the strongest at 35℃(increase by 18.8%),while the POD activity of P.quatuordecimpunctata continued to decrease The T-AOC of H.variegate increased gradually with the increase of temperature,while the T-AOC of P.quatuordecimpunctata decreased significantly at 38℃(decreased by 9.90%).The protein content of the two ladybugs decreased with the increase of temperature,while the protein content of H.variegate was higher than P.quatuordecimpunctata at all temperatures.In conclusion,high temperature stress could affect the antioxidant enzyme system,total antioxidant capacity and protein synthesis of the two ladybugs.However,the physiological response mechanisms of the two ladybugs are different,and these differences may be one of the reasons for the different tolerance to high temperature between the two ladybugs.3.Comparative study on transcriptome of adult of H.variegate and P.quatuordecimpunctata under different temperature stress.Illumina II high-throughput sequencing technology was used to sequence the transcriptomes of H.variegate and P.quatuordecimpunctata at different temperatures(32℃,35℃ and 38℃)for 24 h.A total of 3106 and 3123 differentially expressed genes(DEGs)were identified by H.variegate and P.quatuordecimpunctata under different temperature stresses,respectively.Among them,the number of up-regulated genes in P.quatuordecimpunctata was 2.25 times higher than H.variegate as the temperature rose from 35℃ to 38℃,which indicated that P.quatuordecimpunctata need to mobilize more genes when resisting the high temperature of 38℃.GO enrichment analysis showed that the DEGs involved in the "oxidation-reduction process","catalytic activity" and "oxidoreductase activity" pathways were significantly enriched,and most of the genes involved in these pathways were oxidoreductases.KEGG enrichment analysis showed that there were more DEGs in "metabolic pathway","longevity regulating pathway","antigen processing and presentation"and "amino acid metabolic pathway",and most of the genes involved in these pathways were cytochrome P450 gene family and heat shock protein Hsp70 gene family.The transcriptome analysis data were reliable by quantitative real-time PCR(qRT-PCR),indicating that these genes play a very important role in the process of resistance to high temperature stress in ladybug.Although these DEGs were enriched in both ladybugs,the higher expression times and numbers of DEGs in H.variegate may be the fundamental reason for the different adaptation of the two ladybugs to high temperatures.4.Functional validation of heat shock proteins 70(Hsp70)in adults of H.variegata and P.quatuordecimpunctata.The function of heat shock protein 70 family(H-Hsp70-01,H-Hsp68,and P-Hsp70)in H.variegata and P.quatuordecimpunctata during high temperature stress was explored by microinjection of RNAi.The results of interference efficiency showed that the expression levels of the all target genes were significantly lower than the control(without dsRNA injection)after being interfered for 24h,48h and 72h.When the heat shock protein 70 family genes were successfully interfered,the survival rate,longevity and fecundity of the H.variegata adults(H-Hsp70-01 and H-Hsp68)and P.quatuordecimpunctata adults(P-Hsp70)were significantly lower than the control group(without dsRNA injection)at all experimental temperatures(32℃,35℃,and 38℃),and were more significant at 35℃ and 38℃.Our studies shown that heat shock protein Hsp70 family genes(H-Hsp70-01,H-Hsp68 and P-Hsp70)play a specific role in the resistance of H.variegata and P.quatuordecimpunctata to high temperature stress.Our results provided a scientific basis for analyzing the differences in geographical distribution between H.variegate and P.quatuordecimpunctata in Xinjiang,contributed to a deep understanding of the seasonal dynamics of ladybugs in cotton fields in Xinjiang,as well as provided an important reference for the development of strategies for artificial propagation,indoor conservation and field utilization of natural enemies. |
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