RNA sequencing reveals differential thermal regulation mechanisms between sexes of Glanville fritillary butterfly in the Tianshan Mountains, China


The Glanville fritillary butterfly (Melitaea cinxia; Nymphalidae) has been extensively studied as a model species in metapopulation ecology. We investigated in the earlier studies that female butterflies exhibit higher thermal tolerance than males in the Tianshan Mountains of China. We aim to understand the molecular mechanism of differences of thermal responses between sexes. We used RNA-seq approach and performed de novo assembly of transcriptome to compare the gene expression patterns between two sexes after heat stress. All the reads were assembled into 84,376 transcripts and 72,701 unigenes. The number of differential expressed genes (DEGs) between control and heat shock samples was 175 and 268 for males and females, respectively. Heat shock proteins genes (hsps) were up-regulated in response to heat stress in both males and females. Most of the up-regulated hsps showed higher fold changes in males than in females. Females expressed more ribosomal subunit protein genes, transcriptional elongation factor genes, and methionine-rich storage protein genes, participating in protein synthesis. It indicated that protein synthesis is needed for females to replace the damaged proteins due to heat shock. In addition, aspartate decarboxylase might contribute to thermal tolerance in females. These differences in gene expression may at least partly explain the response to high temperature stress, and the fact that females exhibit higher thermal tolerance.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  1. 1.

    Clarke A (2003) Costs and consequences of evolutionary temperature adaptation. Trends Ecol Evol 18:573–581

    Article  Google Scholar 

  2. 2.

    Angilletta MJ, Niewiarowski PH, Navas CA (2002) The evolution of thermal physiology in ectotherms. J Therm Biol 27:249–268

    Article  Google Scholar 

  3. 3.

    Hoffmann AA, Anderson A, Hallas R (2002) Opposing clines for high and low temperature resistance in Drosophila melanogaster. Ecol Lett 5:614–618

    Article  Google Scholar 

  4. 4.

    Zeilstra I, Fischer K (2005) Cold tolerance in relation to developmental and adult temperature in a butterfly. Physiol Entomol 30:92–95

    Article  Google Scholar 

  5. 5.

    Fallis LC, Fanara JJ, Morgan TJ (2014) Developmental thermal plasticity among Drosophila melanogaster populations. J Evolution Biol 27:557–564

    CAS  Article  Google Scholar 

  6. 6.

    Latimer CAL, Wilson RS, Chenoweth SF (2011) Quantitative genetic variation for thermal performance curves within and among natural populations of Drosophila serrata. J Evolution Biol 24:965–975

    CAS  Article  Google Scholar 

  7. 7.

    Franks SJ, Hoffmann AA (2012) Genetics of climate change adaptation. Annu Rev Genet 46:185–208

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Freitak D, Knorr E, Vogel H, Vilcinskas A (2012) Gender- and stressor-specific microRNA expression in Tribolium castaneum. Biol Lett 8:860–863

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Pezer Z, Ugarkovic D (2012) Satellite DNA-associated siRNAs as mediators of heat shock response in insects. RNA Biol 9:587–595

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Neven LG (2000) Physiological responses of insects to heat. Postharvest Biol Technol 21:103–111

    CAS  Article  Google Scholar 

  11. 11.

    Parsell DA, Lindquist S (1993) The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annu Rev Genet 27:437–496

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Koštál V, Tollarová-Borovanská M (2009) The 70 kda heat shock protein assists during the repair of chilling injury in the insect, Pyrrhocoris apterus. PLoS One 4:e4546

  13. 13.

    Bahrndorff S, Marien J, Loeschcke V, Ellers J (2010) Genetic variation in heat resistance and HSP70 expression in inbred isofemale lines of the springtail Orchesella cincta. Climate Res 43:41–47

    Article  Google Scholar 

  14. 14.

    Štĕtina T, Koštál V, Korbelová J (2015) The role of inducible Hsp70, and other heat shock proteins, in adaptive complex of cold tolerance of the fruit fly (Drosophila melanogaster). PLoS One 10:e0128976

  15. 15.

    Riddoch BJ (1993) The adaptive significance of electrophoretic mobility in phosphoglucose isomerase (PGI). Biol J Linn Soc 50:1–17

    Article  Google Scholar 

  16. 16.

    Neargarder G, Dahlhoff EP, Rank NE (2003) Variation in thermal tolerance is linked to phosphoglucose isomerase genotype in a montane leaf beetle. Funct Ecol 17:213–221

    Article  Google Scholar 

  17. 17.

    Hughes JM, Zalucki MP (1993) The relationship between the Pgi locus and the ability to fly at low-temperatures in the monarch butterfly Danaus plexippus. Biochem Genet 31:521–532

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Sørensen JG, Loeschcke V, Kristensen TN (2009) Lessons from the use of genetically modified Drosophila melanogaster in ecological studies: Hsf mutant lines show highly trait-specific performance in field and laboratory thermal assays. Funct Ecol 23:240–247

    Article  Google Scholar 

  19. 19.

    Udaka H, Percival-Smith A, Sinclair BJ (2013) Increased abundance of Frost mRNA during recovery from cold stress is not essential for cold tolerance in adult Drosophila melanogaster. Insect Mol Biol 22:541–550

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Anderson AR, Collinge JE, Hoffmann AA, Kellett M, McKechnie SW (2003) Thermal tolerance trade-offs associated with the right arm of chromosome 3 and marked by the hsr-omega gene in Drosophila melanogaster. Heredity 9:195–202

    Article  CAS  Google Scholar 

  21. 21.

    Crawford JE, Guelbeogo WM, Sanou A, Traore A, Vernick KD, Sagnon N, Lazzaro BP (2010) De Novo transcriptome sequencing in Anopheles funestus using Illumina RNA-Seq technology. PLoS One 5:e1420212

    Google Scholar 

  22. 22.

    Xu P, Liu ZW, Fan XQ, Gao J, Zhang X, Zhang XG, Shen XL (2013) De novo transcriptome sequencing and comparative analysis of differentially expressed genes in Gossypium aridum under salt stress. Gene 525:26–34

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Vogel H, Badapanda C, Knorr E, Vilcinskas A (2014) RNA-sequencing analysis reveals abundant developmental stage-specific and immunity-related genes in the pollen beetle Meligethes aeneus. Insect Mol Biol 23:98–112

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Ehrlich PR, Hanski I (2004) On the wings of checkerspots: a model system for population biology. Oxford University Press, New York

    Google Scholar 

  25. 25.

    Wahlberg N, Saccheri I (2007) The effects of pleistocene glaciations on the phylogeography of Melitaea cinxia (Lepidoptera: Nymphalidae). Eur J Entomol 104:675–684

    CAS  Article  Google Scholar 

  26. 26.

    Zhou Y, Cao YD, Chen HQ, Long Y, Yan FM, Xu CR, Wang RJ (2012) Habitat utilization of the Glanville fritillary butterfly in the Tianshan Mountains, China, and its implication for conservation. J Insect Conserv 16:207–214

    Article  Google Scholar 

  27. 27.

    Zhou Y, Xu C, Chen HQ, Zhang DD, Long Y, Yan FM, Xu CR, Wang RJ (2013) Different male mate location behaviour of the Glanville fritillary butterfly in different landscapes in the Tianshan Mountains, northwestern China. Entomol Fennica 24:129–139

    Google Scholar 

  28. 28.

    Luo SQ, Wong SC, Xu C, Hanski I, Wang RJ, Lehtonen R (2014) Phenotypic plasticity in thermal tolerance in the Glanville fritillary butterfly. J Therm Biol 42:33–39

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Luo SQ, Ahola V, Shu C, Xu CR, Wang RJ (2015) Heat shock protein 70 gene family in the Glanville fritillary butterfly and their response to thermal stress. Gene 556:132–141

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, Couger MB, Eccles D, Li B, Lieber M (2013) De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc 8:1494–1512

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Ahola V, Lehtonen R, Somervuo P, Salmela L, Koskinen P, Rastas P, Välimäki N, Paulin L, Kvist J, Wahlberg N, Tanskanen J, Hornett EA, Ferguson LC, Luo S, Cao Z, de Jong MA, Duplouy A, Smolander OP, Vogel H, McCoy RC, Qian K, Chong WS, Zhang Q, Ahmad F, Haukka JK, Joshi A, Salojärvi J, Wheat CW, Grosse-Wilde E, Hughes D, Katainen R, Pitkänen E, Ylinen J, Waterhouse RM, Turunen M, Vähärautio A, Ojanen SP, Schulman AH, Taipale M, Lawson D, Ukkonen E, Mäkinen V, Goldsmith MR, Holm L, Auvinen P, Frilander MJ, Hanski I (2014) The Glanville fritillary genome retains an ancient karyotype and reveals selective chromosomal fusions in Lepidoptera. Nat Commun 5:4737–4737

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Li W, Godzik A (2006) Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22:1658–1659

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Bo L, Dewey CN (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12:323

    Article  CAS  Google Scholar 

  34. 34.

    Götz S, García-Gómez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ, Robles M, Talón M, Dopazo J, Conesa A (2008) High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res 36:3420–3435

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Chen X, Mao XZ, Huang JJ, Yang D, Wu JM, Shan D, Lei K, Ge G, Li CY, Wei LP (2011) KOBAS 20: a web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Res 39:W316–W322

    Article  CAS  Google Scholar 

  36. 36.

    Ye J, Fang L, Zheng H, Zhang Y, Chen J, Zhang Z, Wang J, Li S, Li R, Bolund L (2006) WEGO: a web tool for plotting GO annotations. Nucleic Acids Res 34:W293–W292

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Schoville SD, Barreto FS, Moy GW, Wolff A, Burton RS (2012) Investigating the molecular basis of local adaptation to thermal stress: population differences in gene expression across the transcriptome of the copepod Tigriopus californicus. BMC Evol Biol 12:170

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Gleason LU, Burton RS (2015) RNA-seq reveals regional differences in transcriptome response to heat stress in the marine snail Chlorostoma funebralis. Mol Ecol 24:610–627

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Jesus TF, Grosso AR, Almeida-Val VMF, Coelho MM (2016) Transcriptome profiling of two Iberian freshwater fish exposed to thermal stress. J Therm Biol 55:54–61

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Li HB, Shi L, Lu MX, Wang JJ, Du YZ (2011) Thermal tolerance of Frankliniella occidentalis: Effects of temperature, exposure time, and gender. J Therm Biol 36:437–442

    Article  Google Scholar 

  41. 41.

    Blanckenhorn WU, Gautier R, Nick M, Puniamoorthy N, Schäfer MA (2014) Stage- and sex-specific heat tolerance in the yellow dung fly Scathophaga stercoraria. J Therm Biol 46:1–9

    Article  PubMed  Google Scholar 

  42. 42.

    Wang H, Fang Y, Wang LP, Zhu WJ, Ji HP, Wang HY, Xu SQ, Sima YH (2014) Transcriptome analysis of the Bombyx mori fat body after constant high temperature treatment shows differences between the sexes. Mol Biol Rep 41:6039–6049

    CAS  Article  PubMed  Google Scholar 

  43. 43.

    Feder ME, Hoffmann GE (1999) Heat shock proteins, molecular chaperons, and the stress response, evolutionary and ecological physiology. Annu Rev Physiol 61:243–282

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Hoffmann AA, Sorensen JG, Loeschcke V (2003) Adaptation of Drosophila to temperature extremes: bringing together quantitative and molecular approaches. J Therm Biol 28:175–216

    Article  Google Scholar 

  45. 45.

    Sørensen JG, Kristensen TN, Loeschcke V (2003) The evolutionary and ecological role of heat shock proteins. Ecol Lett 6:1025–1037

    Article  Google Scholar 

  46. 46.

    Huang LH, Chen B, Kang L (2007) Impact of mild temperature hardening on thermal tolerance, fecundity, and Hsp gene expression in Liriomyza huidobrensis. J Insect Physiol 53:1199–1205

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    Yu H, Wa, FH, Guo JY (2012) Different thermal tolerance and hsp gene expression in invasive and indigenous sibling species of Bemisia tabaci. Biol Invasions 14:1587–1595

    Article  Google Scholar 

  48. 48.

    Dahlgaard J, Loeschcke V, Michalak P, Justesen J (1998) Induced thermotolerance and associated expression of the heat-shock protein Hsp70 in adult Drosophila melanogaster. Funct Ecol 12:786–793

    Article  Google Scholar 

  49. 49.

    Bahrndorff S, Maien J, Loeschcke V, Ellers J (2009) Dynamics of heat-induced thermal stress resistance and Hsp70 expression in the springtail, Orchesella cincta. Funct Ecol 23:233–239

    Article  Google Scholar 

  50. 50.

    Shen Y, Gu J, Huang LH, Zheng SC, Liu L, Xu WH, Feng QL, Kang L (2011) Cloning and expression analysis of six small heat shock protein genes in the common cutworm, Spodoptera litura. J Insect Physiol 57:908–914

    CAS  Article  PubMed  Google Scholar 

  51. 51.

    Liu QN, Zhu BJ, Dai LS, Fu WW, Lin KZ, Liu CL (2013) Overexpression of small heat shock protein 21 protects the Chinese oak silkworm Antheraea pernyi against thermal stress. J Insect Physiol 59:848–854

    CAS  Article  PubMed  Google Scholar 

  52. 52.

    Martin-Folgar R, de la Fuente M, Morcillo G, Martinez-Guitarte JL (2015) Characterization of six small HSP genes from Chironomus riparius (Diptera, Chironomidae): Differential expression under conditions of normal growth and heat-induced stress. Comp Biochem Phys 188:76–86

    CAS  Article  Google Scholar 

  53. 53.

    Advani NK, Kenkel CD, Davies SW, Parmesan C, Singer MC, Matz M (2016) Variation in heat shock protein expression at the latitudinal range limits of a widely-distributed species, the Glanville fritillary butterfly (Melitaea cinxia). Physiol Entomol 41:241–248

  54. 54.

    Chen B, Kayukawa T, Monteiro M, Ishikawa Y (2005) The expression of the HSP90 gene in response to winter and summer diapause and thermal stress in the onion maggot, Delia antiqua. Insect Mol Biol 14:697–702

    CAS  Article  PubMed  Google Scholar 

  55. 55.

    Zhang QR, Denlinger DL (2010) Molecular characterization of heat shock protein 90, 70 and 70 cognate cDNAs and their expression patterns during thermal stress and pupal diapause in the corn earworm. J Insect Physiol 56:138–150

    CAS  Article  PubMed  Google Scholar 

  56. 56.

    Jiang XF, Zhai HF, Wang L, Luo LZ, Sappington TW, Zhang, L (2012) Cloning of the heat shock protein 90 and 70 genes from the beet armyworm, Spodoptera exigua, and expression characteristics in relation to thermal stress and development. Cell Stress Chaperon 17:67–80

    CAS  Article  Google Scholar 

  57. 57.

    Chen H, Xu XL, Li YP, Wu JX (2014) Characterization of heat shock protein 90, 70 and their transcriptional expression patterns on high temperature in adult of Grapholita molesta (Busck). Insect Sci 21:439–448

  58. 58.

    Rinehart JP, Hayward SAL, Elnitsky MA, Sandro LH, Lee RE, Denlinger DL (2006) Continuous up-regulation of heat shock proteins in larvae, but not adults, of a polar insect. Proc Natl Acad Sci USA 103:14223–14227

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Ittiprasert W, Knight M. (2012) Reversing the resistance phenotype of the Biomphalaria glabrata snail host Schistosoma mansoni infection by temperature modulation. PLoS Pathog 8:363–363

  60. 60.

    Grandbastien MA (2015) LTR retrotransposons, handy hitchhikers of plant regulation and stress response. BBA Biomembr 1849:403–416

    CAS  Google Scholar 

  61. 61.

    Telonis-Scott M, Clemson AS, Johnson TK, Sgro CM (2014) Spatial analysis of gene regulation reveals new insights into the molecular basis of upper thermal limits. Mol Ecol 23:6135–6151

    CAS  Article  PubMed  Google Scholar 

  62. 62.

    Saenko SV, Jeronimo MA, Beldade P (2012) Genetic basis of stage-specific melanism: a putative role for a cysteine sulfinic acid decarboxylase in insect pigmentation. Heredity 108:594–601

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Dai FY, Qiao L, Cao C, Liu XF, Tong XL, He SZ, Hu H, Zhang L Wu SY, Tan D, Xiang ZH, Lu C (2015) Aspartate decarboxylase is required for a normal pupa pigmentation pattern in the silkworm, Bombyx mori. Sci Rep 5:10885

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  64. 64.

    Wittkopp PJ, Carroll SB, Kopp A (2003) Evolution in black and white: genetic control of pigment patterns in Drosophila. Trends Genet 19:495–504

    CAS  Article  PubMed  Google Scholar 

  65. 65.

    Arakane Y, Lomakin J, Beeman RW, Muthukrishnan S, Gehrke SH, Kanost MR, Kramer KJ (2009) Molecular and functional analyses of amino acid decarboxylases involved in cuticle tanning in Tribolium castaneum. J Biol Chem 284:16584–16594

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  66. 66.

    Clusella Trullas S, van Wyk JH, Spotila, JR (2007) Thermal melanism in ectotherms. J Therm Biol 32:235–245

    Article  Google Scholar 

  67. 67.

    True JR (2003) Insect melanism: the molecules matter. Trends Ecol Evol 18:640–647

    Article  Google Scholar 

  68. 68.

    Brisson JA, Toni DCD, Duncan I, Templeton AR (2005) Abdominal pigmentation variation in Drosophila polymorpha: geographic variation in the trait, and underlying phylogeography. Evolution 59:1046–1059

    CAS  Article  PubMed  Google Scholar 

  69. 69.

    Välimäki P, Kivelä SM, Raitanen J, Pakanen VM, Vatka E, Mäenpää MI, Keret N, Tammaru T (2015) Larval melanism in a geometrid moth: promoted neither by a thermal nor seasonal adaptation but desiccating environments. J Anim Ecol 84:817–828

    Article  PubMed  Google Scholar 

  70. 70.

    Hopkins TL, Kramer KJ (1992) Insect cuticle sclerotization. Annu Rev Entomol 37:273–302

    CAS  Article  Google Scholar 

  71. 71.

    Wittkopp PJ, Beldade P (2009) Development and evolution of insect pigmentation: genetic mechanisms and the potential consequences of pleiotropy. Semin Cell Dev Biol 20:65–71

    CAS  Article  PubMed  Google Scholar 

  72. 72.

    Marten JH (2013) Nature’s inordinate fondness for metabolic enzymes: why metabolic enzyme loci are so frequently targets of selection. Mol Ecol 22:5743–5764

    Article  CAS  Google Scholar 

  73. 73.

    Phillips AM, Smart R, Strauss R, Brembs B, Kelly LE (2005) The Drosophila black enigma: the molecular and behavioural characterization of the black 1 mutant allele. Gene 351:131–142

    CAS  Article  PubMed  Google Scholar 

  74. 74.

    Pérez MM, Schachter J, Berni J, Quesada-Allué LA (2010) The enzyme NBAD-synthase plays diverse roles during the life cycle of Drosophila melanogaster. J Insect Physiol 56:8–13

    Article  CAS  PubMed  Google Scholar 

  75. 75.

    Schachter J, Pérez MM, Quesada-Allué LA (2007) The role of N-β-alanyldopamine synthase in the innate immune response of two insects. J Insect Physiol 53:1188–1197

    CAS  Article  PubMed  Google Scholar 

Download references


This work was funded by the Grants 30470286, 30670332 and the China-Finland Collaboration Project 30211130505 from the National Natural Science Foundation of China, the project 6082012 supported by Beijing Natural Science Foundation. We thank BIOPIC for RNA-seq sequencing and Center of Bioinformatics of Peking University for providing the server. We also thank Fuchou Tang, Xiaomeng Liu, Yang Ding and Chao Zhang from Peking University, and Xin Zhou and Shanlin Liu from BGI, who provided technical support in the transcriptome analysis. In addition, we thank Yuping Meng, Zijuan Cao and Jianing Yang for butterfly rearing.

Author information



Corresponding author

Correspondence to Rongjiang Wang.

Ethics declarations

Conflict of interest

The authors declare no conflicts of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 70 KB)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Lei, Y., Wang, Y., Ahola, V. et al. RNA sequencing reveals differential thermal regulation mechanisms between sexes of Glanville fritillary butterfly in the Tianshan Mountains, China. Mol Biol Rep 43, 1423–1433 (2016). https://doi.org/10.1007/s11033-016-4076-x

Download citation


  • The Glanville fritillary butterfly
  • RNA-seq
  • De novo
  • Thermal tolerance