www.nature.com/scientificreports OPEN Molecular chaperoning helps safeguarding mitochondrial integrity and motor functions in Received: 21 November 2017 Accepted: 6 June 2018 the Sahara silver ant Cataglyphis Published: xx xx xxxx bombycina Quentin Willot 1, Patrick Mardulyn 1, Matthieu Defrance2, Cyril Gueydan 3 & Serge Aron1 The Sahara silver ant Cataglyphis bombycina is one of the world’s most thermotolerant animals. Workers forage for heat-stricken arthropods during the hottest part of the day, when temperatures exceed 50 °C. However, the physiological adaptations needed to cope with such harsh conditions remain poorly studied in this desert species. Using transcriptomics, we screened for the most heat- responsive transcripts of C. bombycina with aim to better characterize the molecular mechanisms involved with macromolecular stability and cell survival to heat-stress. We identifed 67 strongly and consistently expressed transcripts, and we show evidences of both evolutionary selection and specifc heat-induction of mitochondrial-related molecular chaperones that have not been documented in Formicidae so far. This indicates clear focus of the silver ant’s heat-shock response in preserving mitochondrial integrity and energy production. The joined induction of small heat-shock proteins likely depicts the higher requirement of this insect for proper motor function in response to extreme burst of heat-stresses. We discuss how those physiological adaptations may efectively help workers resist and survive the scorching heat and burning ground of the midday Sahara Desert. Temperature plays a key role in protein homeostasis1. Most peptides are stable within a narrow thermal range, and increases or decreases in temperature can cause them to unfold and form denatured aggregates1,2. Such sensitivity likely led to the early evolutionary appearance of the heat-shock response (HSR)3,4. One of the HSR’s main func- tions is to increase macromolecular stability, which helps organisms cope efciently with thermal shifs, as well as oxidative stress, heavy metal contamination, or exposure to toxins5,6. A major component of the HSR is the tran- scriptional response, which is controlled by several factors including the evolutionarily conserved transcriptional activator heat-shock factor 1 (HSF1)7. HSF1 trimerizes upon heat shock and binds to consensus heat-shock ele- ments (HSEs) localized on promotor regions of target genes8. Tis response, triggered by the presence of unfolded proteins, leads to the fast and transient transcription of target genes, such as heat-shock proteins (hsps). Hsps are a large family of molecular chaperones. Teir upregulation and accumulation are associated with thermal har- diness9,10. Terefore, the HSR in general, and especially Hsps production, play a central role in allowing cells to survive deleterious conditions. Using transcriptomics, we examined the predominant molecular level processes involved with macromo- lecular stability and cell survival in the Sahara silver ant, Cataglyphis bombycina, focusing on heat-shock pro- teins. Tis species forages during the hottest part of the day, scavenging the bodies of less tolerant, heat-stricken arthropods11. Workers thus experience harsh conditions: air and ground temperatures can reach as high as 50 °C and 70 °C, respectively11. Te silver ant manages this feat thanks to its remarkable ability to survive elevated body 12 temperatures (CTmax = 53.6 °C) . Previous studies have shown that foragers exhibit high constitutive levels of 1Evolutionary Biology and Ecology, Université Libre de Bruxelles, CP 160/12, Av. F.D. Roosevelt, 50, Brussels, 1050, Belgium. 2Interuniversity Institute of Bioinformatics in Brussels, Université Libre de Bruxelles, Boulevard du Triomphe, Brussels, 1050, Belgium. 3Molecular Biology of the Gene, Université Libre de Bruxelles, Rue des Profs. Jeener et Brachet, 12, Gosselies, 6041, Belgium. Correspondence and requests for materials should be addressed to Q.W. (email: [email protected]) SCIENTIFIC REPORTS | (2018) 8:9220 | DOI:10.1038/s41598-018-27628-2 1 www.nature.com/scientificreports/ 22 21 20 19 18 s 17 16 15 14 13 transcript12 of 11 10 9 8 Number7 6 5 4 3 2 1 0 Molecular chaperones --NA-- Hsp90 Co-chaperones Signal transduction Figure 1. Twenty-one transcripts matched up with heat-shockL proteinsipid metabolis and molecularm co-chaperones (31%). Smaller groups of transcripts (<10% each) matched up with proteins with roles ranging from cell-signal transduction to sarcomere myoflament organization. Translation initiation Functional classifcation of the 67 transcripts with strong and consistent heat-induced expression. Oxidative stress response heat-shock cognate 70 (Hsc70) acclimate. However, a deeper understanding of mechanismsUbiquitin- involvedprotea with thesom abilitye degra of cellsdati toon survivepathwa heaty stress while maintaining high metabolic requirements associated with foraging is still lacking in the silver ant. Our aim in this study was to gain a better understanding of the molecular response underlying C. bombycinaChromatin’s abilityremode to ling survive such elevated body temperatures for short periods of time. Calcium transport Results Identification of heat-induced transcripts. Function ofR matchingNA post proteins-transcriptional modifications between 4 groups of heat-stressed (4 tive transcripts (including isoforms) were identifed. Afer removing transcripts with low expression levels, 40,988 Metal binding transcripts remained. Of these, 533 displayed a signifcant regulation in response to heat stress (FDR Fig. S1) and were qualifed as diferentially expressed sequences13,14 (DESs). Expression was downregulated for 147 Immune response DESs and upregulated for 386 DESs. Most displayed a high degree ,of suggesting fold change that (FC) the antsbetween can handlethe two sudden condi- heat exposure without needing to tions; there was also marked variance in transcription within the heat-stress treatment. Amino acid metabolism Similarity annotation. Polyol metabolism sequences (87%) with a high degree of homology were retrieved. Annotation was reliable, as most hits had e-values of less than 1e Sarcomere myofilaments integrity scripts.To A better given characterizetranscript was the retained number only and iffunction (i) mean of FC the was genes greater involved than in 2 betweenthe HSR, heat-stressed we further fltered and control the tran - ants; (ii replicates was less than 0.4. We were thus lef with a total of 67 stronglyh; and 45 consistently expressed transcripts. SCIENTIFIC REPORTS | (2018) 8:9220 | DOI:10.1038/s41598-018-27628-2For this transcript subset, the most represented taxa for the best hit of each match were mainly other ant species, particularly those) FDR in the was subfamily less than Formicinae 0.05; and (iii °C) and 4 groups of control workers (25 na ’s phylogenetic history (Fig. S3). −180 Gene ontology annotation. (Fig. S2). When the 533 DESs were queried against the NCBI nr protein database, 466 533 DESs. Te transcripts were distributed across the three GO-classifcation domains: cellular component (GOWe performed a differential gene expression analysis levels 5–8), biological process (GO levels 4–8), and molecular function (GO levels 3–5) (Fig. S4). co-chaperonesFify-fve (21of the transcripts; 67 transcripts 32%; matched Fig. up with various types of proteins, most commonly Hsps and molecular up with proteins with roles ranging from HSP90 co-chaperones involved in cell division to chromatin remod- elling and sarcomere myoflament organization. Results, including the FC in expression, are depicted in Fig. Complete heat-map of heat-induced transcripts is displayed in Fig. S5. Molecular chaperones involved in the heat-shock response. ) the relative standard deviation (RSD) of expression between biological ated with either molecular chaperones or with co-chaperones involved in protein folding. Of the 67 strongly °C, 4 Based on sequence homology, GO terms could be assigned to 393 (73%) of the h). A total of 301,363 puta- , such as Camponotus foridanus 1). Notably, smaller groups of transcripts (less than 10% each) matched < 0.05; , a result that refects C. bombyci- Of the 533 DESs, 36 were associ- 2. 2 www.nature.com/scientificreports/ Fold Change (log2) −100 10 20 30 40 50 60 6 5 Lipidmetabolis 4 Signaltransductio 3 Hsp90Co−chaperones 2 Nomatc 1 Molecularchaperones 8 Ubiquitin−Proteasomedegradationpath 7 Oxidativ 16 Sarcomere 15 14 Aminoacidmetabolis 13 Immunerespons 12 Metalbinding heat shock 70 kDa cognate 3 11 RNApost−transc 10 Calciumtranspor 9 Chromatinremodelin Tr Po heat shock 70 kDa cognate 4 anslation initiatio heat shock 70 kDa cognate 4 ly ol metabolis heat shock 70 kDa cognate 4 e stressresponse h by highlighting a specifc lineage for which this ratio is signifcantly higher than the background ratio estimated for for estimated ratio thebackground higher than signifcantly is which this ratio for specifc a lineage highlighting by Figure 2 Figure 16: proteins involved in the sarcomere organization. inthe sarcomere involved 16: proteins metabolism; inthe polyol involved 15:proteins metabolism; inthe amino-acid involved 14:Proteins response; inthe immune involved 13:Proteins proteins; binding 12:Metal proteins; 11:RNA-modifying proteins; 10:Calcium transport proteins; remodeling 9:Chromatin pathway; degradation the ubiquitin-proteasome with involved 8:Proteins proteins; response stress 7:Oxidative factors; initiation 6:Translation enzymes; metabolism 5:Lipid 4:Cell proteins; signal transduction