Life Table,Transcriptomic,and Metabolomic Analyses Of Mythimna Separata At Different Constant And Natural Fluctuating Temperatures

The oriental armyworm Mythimna separata(Walker)is an important migratory pest insect species in East Asia.Temperature plays a decisive role on its seasonal distribution.Previous researchers have studied the effects of constant temperatures on the parameters of growth,development and oviposition.However,the factors that influence are not restricted to absolute value of temperature,but involve the variation tendency of temperature.Diurnal and seasonal cycles of temperature exist in nature.Some researchers have found that development rates were different between insects in constant temperature and in diurnal fluctuating temperature but with same average daily temperature.Thus it is indispensable to study the effects of changed temperature on the life history traits.It was studied the effects of laboratory constant temperatures,pupal-alternating temperatures and outdoor fluctuating temperatures on these parameters based on age-stage-specific two-sex life table analyses.Laboratory constant and pupal-alternating temperatures were set as: 20°C,25°C,30°C,20-25°C,25-20°C,and 30°C.The outdoor life table studies were lasted from April 15 th to October 17 th,2018.The four periods,spring,early-summer,mid-summer,and autumn,corresponds to the outbreak regularity of this pest in Central Shaanxi Plain.Then we sequenced the transcriptomes of two day old adult oriental armyworms that were reared at three constant temperatures(20°C,25°C and 30°C)and under outdoor fluctuating environment in different seasons to explore the gene expression patterns in different thermal conditions.At last,the metabolomes of adult M.separata in early summer and autumn were detected by LC-MS technique to find out thermal-responding biomarkers and to explain their history strategy at the view of metabolism.The main results are as follow:1.Life table studies at different constant temperatures and pupal-alternating temperaturesA lower survival rate was observed before 3rd larval instar at 20°C(63.0%)and 20-25°C(70.1%),and an extremely low survival rate after pupation at 30°C(20.6%).Durations in immature stages were shortened with an increasing temperature.The adult pre-oviposition period was shortest at 25°C(2.69 d),and was delayed at low suitable temperature or high temperature before pupation,after pupation,or in the whole developmental stages.Both low suitable and high temperature decreased lifetime fecundity,net reproductive rate and the intrinsic rate of increase,with variability observed across developmental duration and stage during non-optimal temperature.2.Life table studies at natural fluctuating temperatures in different seasonsThe duration of immature stage was significantly shorter in early summer and mid-summer than in spring and autumn.Mortality in 5th larval instar,6th larval instar,and pupal stage was the highest in mid-summer.Fecundity in spring and early summer was larger than in the two later seasons.The intrinsic rate of increase and the finite rate of increase was the highest in early summer,followed by spring,and was the lowest in mid-summer.The net reproductive rate in autumn was lower than in spring and early summer,but higher than in mid-summer.3.Transcriptomic analyses of adult M.separata at different constant temperatures.After quality control,de nove assembly,and redundant reads removing,102078 transcripts were obtained.22911 unigenes were annotated to the non-redundant(NR)database,13465 unigenes to Uniprot/Swiss-Prot database,and 22922 unigenes to Uniprot_ref90 database.Differential gene expression(DEGs)analyses showed that there were 333 downregulated genes and 1588 upregulated genes at 20°C versus 25°C,and 1096 downregulated and 875 upregulated genes at 30°C versus 25°C.GO and KEGG enrichment analyses of DEGs revealed several functional terms related to carbohydrate metabolism,energy metabolism,and xenobiotics metabolism.DEGs involved in glycolysis/gluconeogenesis,the citrate cycle,oxidative phosphorylation pathways,and the composition of myofilaments were significantly downregulated,while many heat shock protein genes(HSPs)and genes in the ubiquitin-mediated proteasome pathway were upregulated at 30 °C.Many cytochrome P450 monooxygenase genes(CYPs)in clan 3 were upregulated at 20 °C,while two genes involved in ecdysteroid biosynthesis,CYP302A1 and CYP315A1,were upregulated at 30 °C.4.Transcriptomic analyses of adult M.separata at outdoor fluctuating temperatures.After de novo assembling and redundant reads removing,272266 transcripts were obtained.82182 unigenes were annotated to NR database,43547 unigenes to Uniprot/Swiss-Prot database,83662 unigenes to Uniprot_ref90 database,50739 unigenes with GO annotation,and 13565 unigenes with KEGG annotation.DEGs analyses showed obviously different gene expression patterns in different seasons.Most DEGs were observed in spring individuals versus in mid-summer individuals with 3846 downregulated genes and 2288 upregulated genes,and least DEGs in early summer individuals versus in autumn individuals with 610 downregulated genes and 842 upregulated genes.GO and KEGG enrichment analyses suggested that many functional terms related to carbohydrate metabolism,energy metabolism,lipid metabolism,amino acid metabolism and xenobiotics metabolism.A large amount of DEGs in glycolysis/gluconeogenesis,in the citrate cycle,and in oxidative phosphorylation pathways were expressed highest in spring,and relatively low in early summer and mid-summer.A certain amount of DEGs fatty acid metabolism were expressed higher in spring and autumn than in early summer and mid-summer.Some heat shock protein genes and proteolytic enzymes genes were highly expressed in early summer and summer,and lowly expressed in spring and autumn.5.Untargeted LC-MS metabolomic analyses of adult M.separata in early summer and in autumn outdoor fluctuating temperaturesPCA and PLS-DA showed a good sample repeatability of metabolome results.12396 features were detected in positive mode,with 1683 downregulated and 1311 upregulated features in autumn than in early summer.6679 features were detected in negative mode,with 1061 features downregulated and 635 genes upregulated.Combined with the annotation results from METLIN metabolites database,it was found that the amounts of carbohydrates,polyols,and intermediate products in TCA cycle and pentose phosphate pathway were higher in autumn individuals.The content of many saturated or unsaturated fatty acids,and free amino acids except serine were higher in early summer individuals.Juvenile hormone III and its degraded products were also higher in early summer individual.