Submitted 26 August 2020Accepted 29 October 2020Published 20 November 2020

Corresponding authorShou-Min Fang,fangshoumin@126.com

Academic editorKenta Nakai

Additional Information andDeclarations can be found onpage 16

DOI 10.7717/peerj.10393

Copyright2020 Fang

Distributed underCreative Commons CC-BY 4.0

OPEN ACCESS

Genome-wide identification and analysisof the thiolase family in insectsShou-Min FangKey Laboratory of Southwest China Wildlife Resources Conservation (Ministry of Education), China WestNormal University, Nanchong, Sichuan, ChinaCollege of Life Science, China West Normal University, Nanchong, Sichuan, China

ABSTRACTThiolases are important enzymes involved in lipid metabolism in both prokaryotesand eukaryotes, and are essential for a range of metabolic pathways, while, little isknown for this important family in insects. To shed light on the evolutionary modelsand functional diversities of the thiolase family, 137 thiolase genes were identified in20 representative insect genomes. They were mainly classified into five classes, namelycytosolic thiolase (CT-thiolase), T1-thiolase, T2-thiolase, trifunctional enzyme thiolase(TFE-thiolase), and sterol carrier protein 2 thiolase (SCP2-thiolase). The intronnumberand exon/intron structures of the thiolase genes reserve large diversification. Subcellularlocalization prediction indicated that all the thiolase proteins were mitochondrial,cytosolic, or peroxisomal enzymes. Four highly conserved sequence fingerprints werefound in the insect thiolase proteins, including CxS-, NEAF-, GHP-, and CxGGGxG-motifs. Homology modeling indicated that insect thiolases share similar 3D structureswith mammals, fishes, and microorganisms. In Bombyx mori, microarray data andreverse transcription-polymerase chain reaction (RT-PCR) analysis suggested that somethiolases might be involved in steroid metabolism, juvenile hormone (JH), and sexpheromone biosynthesis pathways. In general, sequence and structural characteristicswere relatively conserved among insects, bacteria and vertebrates, while different classesof thiolases might have differentiation in specific functions and physiological processes.These results will provide an important foundation for future functional validation ofinsect thiolases.

Subjects Bioinformatics, Entomology, Genomics, Molecular Biology, ZoologyKeywords Insect, Thiolases, Phylogeny, Gene structure, Subcellular localization, Expressionpatterns

INTRODUCTIONThiolases are ubiquitous enzymes that play important roles in lipid-metabolizing pathways(Thompson et al., 1989; Igual et al., 1992; Pereto, Lopez-Garcia & Moreira, 2005). There aretwo major kinds of thiolases based on the direction of the catalytic reaction (Masamuneet al., 1989; Modis & Wierenga, 2000). One is degradative thiolase I (3-ketoacyl-CoAthiolase, E.C. 2.3.1.16), which catalyzes the thiolytic cleavage of medium- to long-chainunbranched 3-oxoacyl-CoAs (from 4 to 22 carbons) into acetyl-CoA and a fatty acyl-CoA(Fig. S1A) (Clinkenbeard et al., 1973; Schiedl et al., 2004; Houten & Wanders, 2010). It ismainly involved in fatty acid β-oxidation and preferentially catalyzes the last step. Theother is biosynthetic thiolase II (acetoacetyl-CoA thiolase, EC2.3.1.9), which is capable of

How to cite this article Fang S-M. 2020. Genome-wide identification and analysis of the thiolase family in insects. PeerJ 8:e10393http://doi.org/10.7717/peerj.10393

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catalyzing the Claisen condensation reaction of twomolecules of acetyl-CoA to acetoacetyl-CoA (Fig. S1B). Thiolase II might be involved in poly beta-hydroxybutyric acid synthesis,steroid biogenesis, etc. (Clinkenbeard et al., 1973).

Based on the function, oligomeric state, substrate specificity, and subcellular localization,six different classes of thiolases (CT, AB, SCP2, T2, T1, and TFE) have been identified inhumans (Fukao, 2002; Mazet et al., 2011; Anbazhagan et al., 2014). In trypanosomatidand bacterial kingdoms, another four classes were identified, including thiolase-likeprotein (TLP), SCP2-thiolase-like protein (SLP), unclassified thiolase (UCT), and TFE-likethiolase (TFEL) (Mazet et al., 2011; Anbazhagan et al., 2014). The CT-thiolase, located inthe cytosol, has a key role in catalyzing the condensation of two molecules of acetyl-CoAto acetoacetyl-CoA, which is the first reaction of the metabolic pathway leading to thesynthesis of cholesterol (Kursula et al., 2005). AB-thiolase and SCP2-thiolase as degradativethiolases occur in peroxisomes (Antonenkov et al., 1997; Antonenkov et al., 1999). T1-, T2-,and TFE-thiolases are mitochondrial degradative enzymes. Except for the degradation ofacetoacetyl-CoA and 2-methyl-acetoacetyl-CoA, T2-thiolase has a biosynthetic functionin the synthesis of acetoacetyl-CoA in ketone body metabolism (Fukao et al., 1997). Ingeneral, AB-, SCP2-, T1-, and TFE-thiolases belong to degradative thiolase I, and CT classis biosynthetic thiolase II. Importantly, T2-thiolase is a bi-functional enzyme with synthesisand degradation activity.

As enzymes responsible for broad pathways, thiolases have been performed thephylogenetic analysis in mycobacteria and functional studies in humans (Mazet et al.,2011; Xia et al., 2019). In insects, several thiolase genes have been characterized (Fujii etal., 2010). In Helicoverpa armigera, an acetoacetyl-CoA thiolase was cloned and performedfunctional analysis (Zhang et al., 2017). It was indicated that the thiolase was involved in theearly step of the juvenile hormone pathway, i.e., mevalonate biosynthesis. One acetoacetyl-CoA thiolase was purified to apparent homogeneity by column chromatography in Bombusterrestris, suggesting that it might be the first enzyme in the biosynthesis of terpenic sexpheromone (Brabcova et al., 2015). Besides, acetyl-CoA can be used as the precursor for denovo biosynthesis of sex pheromones in female moths, and four 3-ketoacyl-CoA thiolasegenes were identified in sex pheromone gland transcriptome of the noctuid mothHeliothisvirescens (Vogel et al., 2010). In total, there are few and scattered studies on insect thiolase,lacking systematic identification and comparative studies.

In this study, we selected 20 representative species from seven insect orders to performgenome-wide identification of the thiolase family proteins. Gene structure, chromosomelocation, and three-dimensional (3D) structure and motif characteristics of proteinswere compared. In addition, Bombyx mori is an important model species for studyingjuvenile hormone and sex pheromone biosynthesis (Matsumoto, 2010; Xia, Li & Feng,2014). Expression profiles of the thiolase genes were detected in various tissues anddevelopmental sex pheromone gland of B. mori. Combining structural characteristics andexpression patterns, the potential functions and involved physiological processes werehypothesized. The present study can help us understand the functional differentiation ofthiolase genes in insects.

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Table 1 Classification of the thiolase genes among insects,H. sapiens andM. tuberculosis.

Organisms Total CT T1 T2 AB TFE TFEL TFEL SCP2 SCP2(type-1) (type-2) (type-1) (type-2)

H. sapiens 6 1 1 1 1 1 1M. tuberculosis 9 1 1 3 1 1 1Lep_B. mori 6 3 1 1 1Lep_P. xuthus 10 2 2 2 1 3Lep_M. sexta 6 3 1 1 1Lep_D. plexippus 5 2 1 1 1Lep_H. melpomene 4 1 1 1 1Lep_P. xylostella 15 1 10 1 2 1Dip_C. quinquefasciatus 6 2 1 1 1 1Dip_A. gambiae 7 1 2 1 2 1Dip_D. melanogaster 6 1 1 1 1 2Col_A. glabripennis 5 2 2 1Col_N. vespilloides 6 1 1 2 1 1Col_T. castaneum 5 1 1 1 1 1Hem_H. halys 5 1 1 1 1 1Hem_A. pisum 12 7 1 1 1 2Hem_C. lectularius 8 2 1 1 2 1 1Hym_D. alloeum 8 2 1 3 1 1Hym_A. mellifera 5 1 1 1 1 1Hym_B. impatiens 8 1 2 2 1 2Pht_P. humanus 6 1 1 2 1 1Iso_Z. nevadensis 4 1 1 1 1

Notes.Lep, Lepidoptera; Dip, Diptera; Col, Coleoptera; Hem, Hemipter; Hym, Hymenoptera; Pht, Phthiraptera; Iso, Isoptera.Thiolase-like protein (TLP) ofM. tuberculosis was not listed in the table, and was not found homologous genes in insects and human.

MATERIALS & METHODSData resourcesIn this study, 20 representative species were selected from Lepidoptera, Hymenoptera,Hemiptera, Diptera, Coleoptera, Phthiraptera, and Isoptera (Table 1). The annotated genesand genomes ofB. moriwere retrieved fromSilkDB v3.0 (https://silkdb.bioinfotoolkits.net).The sequence information of Danaus plexippus and Heliconius melpomene weredownloaded from http://metazoa.ensembl.org/. Manduca sexta was downloadedfrom https://i5k.nal.usda.gov/. The other sequences were retrieved from GenBank(https://www.ncbi.nlm.nih.gov/), including Papilio xuthus, Plutella xylostella, Culexquinquefasciatus, Anopheles gambiae, Drosophila melanogaster, Anoplophora glabripennis,Nicrophorus vespilloides, Tribolium castaneum, Halyomorpha halys, Acyrthosiphon pisum,Cimex lectularius, Diachasma alloeum, Apis mellifera, Bombus impatiens, Pediculushumanus, and Zootermopsis nevadensis.

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Identification of insect thiolase genesThe known thiolase sequences of Homo sapiens and Mycobacterium tuberculosis wereretrieved fromGenBank (Table S1; Anbazhagan et al., 2014) and used as queries to performBLASTP search (E- value

structural similarities among the insect thiolases, all the other structures were comparedwith BmorT2 using magic fit algorithm in Swiss-PdbViewer, respectively. The root-mean-square distance (RMSD) values were calculated to express the structural similarity. Thelower value of RMSD means higher similarity between two structures (Carugo & Pongor,2001).

Reverse transcription-polymerase chain reaction (RT-PCR)The various tissues on day 3 of fifth-instar larvae were dissected in the silkworm. Thesex pheromone glands (PGs) from five individuals were used as one sample at eachdevelopmental stage. All the samples were preserved in RNAlater (Ambion, 98 Austin,USA) and stored at −80 ◦C for RNA isolation. Total RNA was extracted using Trizolreagent (Invitrogen, USA). The first strand of cDNA was synthesized by M-MLV reversetranscriptase following the manufacturer’s instructions (Promega, USA). RT-PCR primerswere listed in Table S2. The silkworm RpL3 gene was used as an internal control forrelative quantitative analysis of RT-PCR. PCRs were performed with the following cyclingparameters: 95 ◦C for 3 min (min), followed by 25 cycles of 30 s (s) at 95 ◦C, 30 s annealing(temperatures listed in Table S2) and 30 s extension (72 ◦C), and a final extension at 72 ◦Cfor 10 min. The amplification products were monitored on 1.5% agarose gels.

RESULTSGenome-wide identification and phylogeny of insect thiolase proteinsTo identify thiolases in insects, human andM. tuberculosis thiolase protein sequences wereused as queries to perform homologous searches in whole genomes. In total, 137 thiolasegenes were identified in 20 insects from seven orders, and the gene numbers of the specieswere ranged from 4 to 15 (Table 1; Table S1). The thiolase protein sequences were used toreconstruct themaximum-likelihood phylogenetic tree (Fig. 1). Based on the nomenclaturerules in humans andM. tuberculosis (Anbazhagan et al., 2014), each thiolase was named ininsects. It was indicated that insect thiolases were grouped 7 classes, namely CT, T1, T2,TFE, SCP2 (type-1), TFEL (type-2), and AB (Table 1; Fig. 1). Relatively, the classification ofinsect thiolases was more similar to that of the human thanM. tuberculosis. Unlike humans,out of 20 insect species, only P. xuthus has gene members in AB class. Interestingly, 5 outof 6 Lepidopteran insects have no CT-thiolase. Furthermore, TFEL (type-2) class was onlydetected in C. lectularius (Hemipter) and bacteriumM. tuberculosis.

To understand the evolutionary mode, gene gain and loss of thiolases were analyzed. Itwas indicated thatmost of the gain and loss events were occurred in a certain species (Fig. 2).Especially, P. xylostella (Lepidoptera), A. pisum (Hemipter), and P. xuthus (Lepidoptera)showed more duplications after or during the formation of the species, resulting in a totalnumber of 15, 12, and 10 genes, respectively. Except for the gene duplication of a singlespecies lineage, the common ancestor of D. alloeum, A. mellifera and B. impatiens showed2 duplications (Fig. 2). This phenomenon was also noted in the clade of Hemipteran H.halys, C. lectularius, and A. pisum. For those recent duplication genes, they were oftenphylogenetically closely related to its ancestral genes (Fig. 1). Conversely, A. mellifera(Hymenoptera), H. halys (Hemipter), and Z. nevadensis (Isoptera) presented 3, 3, and 2

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Figure 1 Phylogenetic tree of insect thiolases using the maximum-likelihood (ML) method. Thethiolases of human andM. tuberculosis were shown by stars and dots, respectively. The bootstrap valueshigher than 50% were dotted on the nodes. Lep: Lepidoptera; Dip: Diptera; Col: Coleoptera; Hem:Hemipter; Hym: Hymenoptera; Pht: Phthiraptera; Iso: Isoptera. The accession numbers following eachgene name were presented. The information ENSANGP within accession numbers of A. gambiae wasomitted.

Full-size DOI: 10.7717/peerj.10393/fig-1

gene losses during speciation, resulting in fewer genes in these species. Generally, gene gainand loss rates are important for understanding the role of natural selection and adaptationin shaping gene family sizes. For the species with more gene expansion, whether theseduplicated genes play roles in adapting to special habitats deserves further study.

Gene structures of insect thiolase genesA comparative analysis of exon-intron structures was conducted for the 137 insect thiolasegenes (Figs. 3A and 3B; Fig. S2A). The insect thiolase genes have a different number ofintrons ranging from 0 to 22. It was indicated that only 12 genes have no intron, and 17

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Culex quinquefasciatus (6)

Bombus impatiens (8)

Diachasma alloeum (8)

Pediculus humanus (6)

Acyrthosiphon pisum (12)

Plutella xylostella (15)

Cimex lectularius (8)

Heliconius melpomene (4)

Homo sapiens (6)

Tribolium castaneum (5)

Zootermopsis nevadensis (4)

Apis mellifera (5)

Nicrophorus vespilloides (6)

Bombyx mori (6)

Manduca sexta (6)

Halyomorpha halys (5)

Mycobacterium tuberculosis (9)

Anoplophora glabripennis (5)

Drosophila melanogaster (6)

Papilio xuthus (10)

Danaus plexippus (5)

Anopheles gambiae (7)

200.0 million years ago (MYA)

6

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66

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Figure 2 Gene gain and loss analysis of the thiolase gene family in insect genomes. The species tree wasobtained from the TimeTree database (http://www.timetree.org/). Gain and loss analysis was conductedby Notung-2.9 software with default parameters. The orange and green vertical bars on branch presentedgene gain and loss, respectively. The number in each node is the gene count. The gene number of eachspecies was presented in brackets.

Full-size DOI: 10.7717/peerj.10393/fig-2

genes have only one intron, accounting for 21.17% in total. The intronless genes weredistributed in T2 (5), SCP2 (type-1) (2), CT (2), AB (2), and TFEL (1). Previous studiesrevealed that introns can delay regulatory response and are selected against in genes whosetranscripts need to be adjusted quickly to meet environmental challenges (Jeffares, Penkett& Bahler, 2008). The intronless thiolase genes and the genes contained fewer introns mightplay important roles in survival for environmental changes. In addition, the intron numberand exon/intron structures of thiolase genes are very different, even the orthologous genesof different species in the same class have a large differentiation. It was suggested thatthe differentiation of the intron number may result in the diversification of thiolase genestructures in insects.

Fang (2020), PeerJ, DOI 10.7717/peerj.10393 7/21

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Hem_ApisCT-1

Dip_DmelSCP2 (type-1)-2

Dip_DmelTFE

Iso_ZnevTFE

Iso_ZnevCT

Col_TcasT1

Lep_BmorT1-1

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Hem_ApisT1

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Lep_HmelSCP2 (type-1)

Lep_HmelT2

Lep_HmelT1

Pht_PhumTFE

Dip_DmelSCP2 (type-1)-1

Hym_AmelCT

Iso_ZnevT1

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Hem_ApisCT-5

Lep_BmorTFE

Hym_AmelTFE

Hem_ApisCT-4

Hym_AmelT2

Pht_PhumCT

Pht_PhumT2-1

Hym_AmelT1

Pht_PhumT1

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Lep_BmorT1-3

Pht_PhumSCP2 (type-1)

Hem_ApisCT-7

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Hem_ApisCT-2

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Col_TcasCT

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Hem_ApisSCP2 (type-1)-2

Pht_PhumT2-2

Dip_DmelT2

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(A) Phylogenetic tree

Figure 3 Exon/intron structure and subcelluar localization analyses. (A) The maximum-likelihoodphylogenetic tree of thiolase proteins from seven representative insects. The bootstrap values higher than50% were dotted on the nodes. (B) Exon/intron structure analysis of the thiolase genes. (C) Subcelluar lo-calization analysis of thiolase proteins. It was predicted by the PSORT II server (https://www.genscript.com/tools/psort). Mit, mitochondrion; Cyt, cytosol; Pox, peroxisome.

Full-size DOI: 10.7717/peerj.10393/fig-3

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Chromosome distribution and gene syntenyIn order to explore the chromosomal distribution of thiolase genes, five representativespecies were analyzed. It was indicated that most of the thiolase genes were randomlydistributed on different chromosomes (Figs. S3A–S3E), for example, six thiolase geneswere scattered on four chromosomes in D. melanogaster (Fig. S3A), which is similar tothiolase genes in the human (Anbazhagan et al., 2014). However, different members of acertain class are often tandem distribution, such as DmelSCP2 (type-1)-1 and DmelSCP2(type-1)-2 in D. melanogaster (Fig. S3A) and BmorT1-1, BmorT1-2, and BmorT1-3 in B.mori (Fig. S3C). Meanwhile, we also detected the distribution of the 10 T1-thiolase genesin P. xylostella and 7 CT-thiolase genes in A. pisum, which were distributed on several smallunassembled scaffolds. Whether they are distributed in tandem, we still need to wait forthe scaffold sequences to be integrated into the corresponding chromosome in future. Ingeneral, tandem duplication might be the main mechanism for enlarging thiolase familyin insects.

The syntenic relationships of thiolase genes were investigated among B. mori, H.melpomene, D. melanogaster, T. castaneum, and A. mellifera because their genomesequences have been assembled into chromosome levels. The results indicated that onlyfour genes exhibited the syntenic relationships between B. mori and H. melpomene, thatis, BmorT2 and HmelT2, BmorTFE and HmelTFE (Fig. S3F). Interestingly, except forthe tandemly duplicated genes, amount of thiolase genes often present orthologousrelationships among insect, human, and M. tuberculosis (Fig. 1). It was suggested thatthiolase family is an ancient gene family. Even in insects, the age of thiolase genedifferentiation is relatively long. Thus, the discovery of fewer syntenic genes impliesthat thiolases might mainly locate in some non-conserved genomic blocks (The HeliconiusGenome Consortium, 2012).

Subcellular localization of thiolase proteinsSubcellular localization refers to the specific location of a certain protein or the expressionproduct of a certain gene in the cell. Protein subcellular localization is closely related toprotein functions (Pereto, Lopez-Garcia & Moreira, 2005;Wang et al., 2014). Only when theprotein is positioned correctly can it perform normal biological functions. In this study,subcellular localization of all the 137 insect thiolase proteins was predicted by PSORTII server (https://www.genscript.com/tools/psort), which were cytosolic, mitochondrial,or peroxisomal enzymes (Fig. 3C; Fig. S2B). Generally, most of the TFE- and T2-thiolaseproteinswere located in themitochondrion, T1- andCT-thiolaseswere cytosolic, and SCP2-thiolases were peroxisomal proteins. Previous studies suggested that the mitochondrialand peroxisomal thiolase proteins were mainly involved in the fatty acid β-oxidationpathway (Pereto, Lopez-Garcia & Moreira, 2005), and cytosolic localization was related tothe biosynthesis of acetoacetyl-CoA (Kursula et al., 2005). However, in a certain class ofthiolase, there are always a few exceptions to the cellular location in some species (Fig. 3C;Fig. S2B), which suggested that its function might have diverged during evolution.

Fang (2020), PeerJ, DOI 10.7717/peerj.10393 9/21

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Conserved domain characteristics and catalytic residuesTo identify the potential domains of insect thiolase proteins (Table S1), it was performedhmmscan analysis in Pfam database. The results indicated that all the thiolases containedThiolase_N and Thiolase_C domains (Fig. 4A). In addition to the thiolase domains,SCP2-thioloase (type-1) has a typical sterol carrier protein 2 (SCP2) domain at C terminal.Unexpectedly, some of the members in TFE, CT, and T2 classes contained a ketoacyl-synt(beta-ketoacyl synthase) domain within Thiolase_N (Table S1). We carefully checked thealignments of hmmscan search. It was found that the E value was around the threshold1e−5, and only about 50 amino acids can be aligned, which are much shorter than 250amino acids of the ketoacyl-synt domain (Pfam ID, PF00109). Thus, thiolases may notcontain the real ketoacyl-synt domain, and just show certain similarities with it (Huang etal., 1998). Therefore, based on the domain characteristics, all the insect thiolase encodinggenes were classified as 2 groups (Fig. 4A).

The conserved sequence blocks of the 20 insects, humans, and M. tuberculosis wereanalyzed (Fig. S4 ; Fig. 4B). The CxS-motif is the most important sequence fingerprint inthe N-terminal domain, which provides the nucleophilic cysteine (Zeng & Li, 2004; Mazetet al., 2011). Except for some incomplete sequences, almost all the thiolases containedthe cysteine residue (Fig. S4 ; Fig. 4B). The histidine of the GHP-motif contributes tothe oxyanion hole of the thioester oxygen (Merilainen et al., 2009). It was indicated thatGHP-motif was highly conserved in insects, humans, andM. tuberculosis (Fig. S4 ; Fig. 4B).The cysteine of CxGGGxG-motif provides the catalytic residue of the active sites. Exceptfor SCP2-thiolases, the catalytic cysteine was retained in almost all the other thiolases (Fig.S4). In addition, the asparagine side chain of the NEAF-motif interacts with importantcatalytic water (Mazet et al., 2011). However, NEAF-motif was replaced by HDCF-motifin all of the SCP2-thiolases (Fig. S4 ; Fig. 4B). Based on the comparison of the sequencefingerprints, it was indicated that the catalytic mechanisms of the insect thiolases might besimilar to that of thiolases from mammals and bacteria.

Molecular modeling of insect thiolasesIn recent years, the crystal structures of some thiolases have been gradually resolved inbacteria, fish, and mammals (Harijan et al., 2013; Kim et al., 2015; Xia et al., 2019). Thehigh sequence similarities (>60%) may help to build more accurate 3D structures for theinsect thiolases (Arnold et al., 2006). Based on homology modeling using SWISS-MODELInteractive Workspace, we found that thiolase sequences within a class were very conservedamong different organisms. For instance, BmorTFE-thiolase and BmorSCP2-thiolase(type-1) shared 67.58% and 61.63% identities with its corresponding modeling templatesfrom human (PDB ID: 6dv2.1.A) and zebrafish (6hrv.2.A), respectively. In this study,the modeling structures of some representative thiolases were presented (Figs. 5A–5F).The structural similarities of the monomeric forms were detected with magic fit in Swiss-PdbViewer (Guex & Peitsch, 1997). The RMSD values were ranged from 0.25 Å to 0.91Å among BmorT2, BmorT1-1, DmelCT, and PxutAB-1, while BmorTFE and BmorSCP2(type-1) shared from 1.12 Å to 2.01 Å with the others (Table S3). It was indicated that

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* * *NEAF GHP CxGGGxGCxS

Thiolase_CThiolase_N SCP2

50 aaT1/T2/AB/CT/TFE/TFEL

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I P I Y N V N N N C S T G Q V D V V E L H D C F A A N P S G G L I A K G H P L Q L G G G M GC P I Y N V N N N C S T G D V D V I E L H D C F S I N P S G G L I S K G H P L Q I G G G L GI P V Y N V N N N C S T G D V Q V V E L H D C F S A N P S G G L I S K G H P L Q L G G G L GI P V Y N V N N N C S T G D V Q V V E L H D C F S A N P S G G L I S K G H P L Q L G G G L GI P I F N V N N N C S T G D V Q V V E L H D C F S A N P S G G L I S K G H P L Q I G G G L GI P I Y N V N N N C S T G Q I D V V E L H D C F A A N P S G G L I A K G H P L Q L G G G M GI P I I N V N N N C A T G D I D V I E L H D C F S T N P S G G L I S K G H P L Q L G G G L GI P I V N V N N N C S T G D F G V I E L H D C F S A N P S G G L I S K G H P L Q I G G G - AT P A T R H E A A C A S G D L D G I E V H D C F P S N P S G G L I - - G H P A - E A G G - GK P V L G I N R L C G S G D V D L I E I N E A F V A N V N G G A T A L G H P A C I G G G Q GK P A L G V N R L C G S G D I D L V E I N E A F A A N V N G G A I A M G H P A C I G G G Q GR P A L G V S K A C G T G D V D L F E I N E V F A S N V N G G A L A F G N P S C - G G G Q GK P S F S L N R L C G S G D I E L I E I N E A F G S N V D G G S I A L G H P A C I G G G Q GK P A L G I N R L C G S G D I D L I E I N E A F A A N V N G G A I A L G H P A C I G G G Q GR P A F L V N R L C G S G D I D L I E I N E A F A A N V D G G A I A L G H P A C I G G G Q GK P V L G V N R L C G S G D V D L I E V R - V Y - - - - - G G Q L S V - H P V C D G G G L GT P A L G V N R L C G S G D V D L V E I N E A F A A N V S G G A I A M G H P A C I G G G Q GR P A L V V N M A C G A G D V D L I E I N E T F A A N V N G G A L A V G H P S C - G G G Q GR P A L A V N L A C G A G G V D L I E I N E T F A A N V N G G A L A V G H P S C - G G G Q GR P A L G V N L A C G A G G V D L I E I N E T F A A N V N G G A L A V G H P S C - G G G Q GR P A L A V N L A C G A G S V D L I E I N E T F A A N V N G G A L A V G H P - - - - - - - -- - - - - - - - - - - - - D V D L V E I N E A F A A N V N G G A I A M G H P A C I G G G Q G- - - - - - - - - C V - - D V D L V E I N E A F A A N V N G G A I A M G H P A C I G G G Q GK P V L G V N R L C G S G D V D L I E I N E A F C A N V N G G A T A L G H P A C I G G G M GH P R - - - - - - - - E S D V D L I E I N E A F C A N V N G G A T A L G H P - - - - - - - -T P A L T I N R L C G S G D M D L V E V N E A F A P N V N G G A I A L G H P A C I G G G Q GT I C T T V N K V C A S G D V A L W E I N E A F S V N V H G G A V S L G H P I C N G G G - GT I C T T V N K V C A S G D I A L W E I N E A F S V N I H G G A V S L G H P I C N G G G - GV C C T T V N K V C S S G D V A M W E V N E A F S L N V H G G A V S I G H P I C N G G G - GT I C T T I N K V C A S G D V A L W E I N E A F S V N V H G G A V S L G H P I C N G G G - GT I C T T I N K V C S S G E V A L W E L N E A F S V N V H G G A V S I G H P I C N G G G - GT P C T T I N K V C A S G D I A M W E V N E A F S L N I N G G A V S L G H P I C N G G G - GV P A L T I N K M C L S G Q L D V V E I N E A F A A N V N G G A I A V G H P L C - G G G Q GT P A Y L I N M L C G S G E V D L Y E L N E A F A A N I N G G A I A L G H P L C I G G G M GV P A Y G I N M L C G S G E V D L Y E L N E A F A A N V N G G A I A L G H P L C I G G G M GV P A Y T I N M L C G S G D V D L Y E L N E A F A V N I T G G A I A L G H P L C I G G G M GV S A I T I N D V C G S G D V D L I E V N E A F A A N V N G G A I A L G H P L C I G G G Q GV P A W S C Q M I C G S G D V D I F E I N E A F A A N I E G G A I A L G H P L C I G G G M GV P L S T V N R Q C S S G D V D I F E I N E A F A S N P L G G A V A L G H P M C I G G - M GT P A H T V T M A C I S S D I D T W E I H E A F A G N K WG G S L S I G H P A C A A G G Q GI P A H T V T M A C I S S D I G V W E I H E A F A G N A WG G S L S I G H P A C A A G G Q GT P A H T V T M A C I S S D I D S W E I H E A F A G N N WG G S L S I G H P A C A A G G Q GT P A H T V T M A C I S S D I A V W E F H E A F A G N N WG G S L S I G H P A C A A G G Q GT P A H T V T M A C I S A D I D T W E I H E A F A G N N WG G S L S I G H P A C A A G G Q GT P A H T V T M A C I S A D I D T W E I H E A F A G N N WG G S L S I G H P A C A A G G Q GT P A H T V T M A C I S A D I D A F E F H E A F S G N N WG G S L S L G H P A C A A G G Q GT P A F D L Q Q A C G T G D F D F Y E I H E A F A S N V N G S S L A A G H P I C A A G G Q GL P G T T V N R Y C S S S D I D L V E I N E A F A V N I S G G A I A L G H P M C V G G G Q GS G G V Q L N R F C A S G D I D L F E L N E A F A S N V N G G A I A M G H P L C I G G G M GV P G V T V D R Q C G S S D I D T V E I N E A F A P N P N G G A I A L G H P M C E G G G T AV G A T T V D C Q C G S G D I D I V E I N E A F A S N V N G G A I A L G H P M C A G G A L GM P Q S L V N R A C L - - D I E Y V D L Y S C F S A T V T G G - L T F G G P A - N G G G V G

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Lep_BmorSCP2 (type-1) Hym_AmelSCP2 (type-1) Dip_DmelSCP2 (type-1)-1Dip_DmelSCP2 (type-1)-2Col_TcasSCP2 (type-1) Lep_PxylSCP2 (type-1) HsapSCP2 (type-1) MtubSCP2 (type-1) MtubSCP2 (type-2) Lep_BmorT1-1 Lep_BmorT1-2 Lep_BmorT1-3 Hym_AmelT1 Dip_DmelT1 Col_TcasT1 Lep_PxylT1-1 Lep_PxylT1-2 Lep_PxylT1-3 Lep_PxylT1-4 Lep_PxylT1-5 Lep_PxylT1-6 Lep_PxylT1-7 Lep_PxylT1-8 Lep_PxylT1-9 Lep_PxylT1-10 HsapT1 Lep_BmorT2 Hym_AmelT2 Dip_DmelT2 Col_TcasT2 Lep_PxylT2 HsapT2 MtubT2 Hym_AmelCT Dip_DmelCT Col_TcasCT Lep_PxylCT HsapCT HsapAB Lep_BmorTFE Hym_AmelTFE Dip_DmelTFE Col_TcasTFE Lep_PxylTFE-2 Lep_PxylTFE-1 HsapTFE MtubTFE MtubTFEL (type-2) MtubTFEL (type-1)-1 MtubTFEL (type-1)-3 MtubTFEL (type-1)-2 MtubTLP

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Figure 4 Conserved domains and fingerprints of insect thiolases compared with humans andbacteriumM. tuberculosis. (A) The conserved domains predicted by hmmscan against Pfamdatabase. Three types of domains were found in insect thiolases, including N-terminal of thiolase(Thiolase_N), C-terminal of thiolase (Thiolase_C), and sterol carrier protein 2 (SCP2). BmorSCP2(type-1) and BmorT1-1 were used to represent the common structures, respectively. (B) Highly conservedsequence blocks containing fingerprints CxS, NEAF, GHP, and CxGGGxG. Three catalytic residuesresponsible for thiolase activity are indicated by stars. The alignement logos were generated by WebLogo(http://weblogo.berkeley.edu/logo.cgi).

Full-size DOI: 10.7717/peerj.10393/fig-4

Fang (2020), PeerJ, DOI 10.7717/peerj.10393 11/21

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Figure 5 Protein homology modeling and 3D structures of insect thiolases. The monomer structures ofsome representative thiolases were shown, including BmorT2 (A), DmelCT (B), PxutAB-1 (C), BmorT1-1 (D), BmorTFE (E), and BmorSCP2 (type-1) (F). The corresponding templates for homology modelingwere PDB ID 6bjb.1.A, 4wyr.1.A, 1afw.1.A, 4wyr.1.A, 6dv2.1.A, and 6hrv.2.A, respectively. The dotted sur-face of the three catalytic residues was presented in (A) to (F) monomer structures. (G) Homo-tetramer ofBmorT1-1. (H) Homo-dimer of BmorSCP2 (type-1).

Full-size DOI: 10.7717/peerj.10393/fig-5

T1, T2, CT, and AB classes share more similar 3D structures than TFE-thiolase and SCP2-thiolase (type-1) (Figs. 5A–5F). This phenomenon is widespread in both humans and M.tuberculosis (Harijan et al., 2013; Anbazhagan et al., 2014). For the quaternary structure,different thiolases also have certain differences. For example, BmorT1-1 and BmorSCP2(type-1) were homo-tetramer and homo-dimer, respectively (Figs. 5G and 5F). The resultsof 3D structural modeling showed that different classes of thiolase genes still present someextent divergence in tertiary or quaternary structures.

SCP2-thiolase (type-1) was widely distributed in insects, mammals, and bacteria (Table1). One single structural gene referred to as the sterol carrier protein x (SCPx) gene encodes

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a full-length protein comprised of 3-oxoacyl-CoA thiolase (known as SCP2-thiolase)and sterol carrier protein 2 (Seedorf et al., 1994; Gallegos et al., 2001). The C-terminalSCP2-domain containing the peroxisomal targeting signal is needed for the targeting offull-length SCPx into the peroxisomes. The SCP2-thiolase and SCP2 protein are producedfrom SCPx via proteolytic cleavage by peroxisomal proteases (Seedorf et al., 1994). Basedon the homology modeling, the tertiary and quaternary structures of mature SCP2-thiolase(type-1) protein were presented in Figs. 5F and 5H, respectively. For insect SCP2-thiolases,the canonical CxGGGxG-motif is also absent, and the NEAF-motif has been replaced byHDCF-motif (Fig. 4B). The previous studies indicated that HDCF-motif might provide thecatalytic cysteine in bacteria, mammals, and fish (Harijan et al., 2013; Kiema et al., 2019).Based on the structural modeling, the cysteine of HDCF-motif is very close to the other twocatalytic sites in protein spatial conformation (Fig. 5F). Therefore, the catalytic cysteine ofthe insect SCP2-thiolases might be not provided by CxGGGxG-motif but HDCF-motif.

Expression profile and potential functional diversityTo understand the potential functional diversity of the insect thiolases, the silkworm, B.mori, was used as a model organism to perform expression profile analysis in the varioustissues and sex pheromone glands (PGs) at different developmental stages. In the silkworm,genome-wide microarray with 22,987 oligonucleotides was designed and surveyed the geneexpression profiles in multiple tissues on day 3 of the fifth-instar larvae (Xia et al., 2007).5 out of 6 thiolase genes were found its corresponding probes (Fig. 6A). The microarraydata indicated that BmorSCP2 (type-1), BmorT2, BmorTFE, and BmorT1-1 have expressionsignals at least one of the 9 tissues. Relatively, BmorT2 and BmorT1-1 showed ubiquitousexpressions. Meanwhile, the expression profiles of the four genes were similar betweenfemales and males, respectively.

To validate the expression profiles of the silkworm thiolase genes, the mixed maleand female tissues were used to perform RT-PCR validation on day 3 of the fifth-instarlarvae (Fig. 6B). In total, 5 out of the 6 thiolase genes presented expression evidence.Relatively, BmorT1-2 and BmorTFE showed predominant expressions in hemocyte andhead, respectively (Fig. 6B), while BmorT1-1 was widely expressed in various tissues. Inaddition, sex pheromone glands of different developmental stages were used to detect theexpressions of thiolase genes (Fig. 6C). Four thiolase genes presented expression signals inthe silkworm PGs. Relatively, BmorT1-1 showed the highest expression on day 8 of pupae.BmorTFE, BmorT2, and BmorSCP2 (type-1) presented expressions at all the developmentalstages. Interestingly, the expression levels of all three genes were declined in the matedfemale PGs (Fig. 6C). These expression analyses might help us understand the functionaldivergence of the thiolase genes in the silkworm.

DISCUSSIONThiolases are widely distributed in all organisms and are essential for a range of metabolicpathways. With the development of sequencing technology, it provides the possibilityfor us to identify and compare insect thiolase at the whole genome level. In this study,137 thiolase genes were identified in the 20 representative species from 7 insect orders

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structural component of cell membranes (Gilbert, Rybczynski & Warren, 2002). Due to thelack of squalene monooxygenase and lanosterol synthase for the synthesis of cholesterol,insects can not autonomously synthesize the 20E precursor (Guo et al., 2009). Alternatively,insects can obtain cholesterol or other sterols from their diet to meet the needs of growthand development. In Spodoptera litura, one sterol carrier protein x (SCPx) gene encodinga sterol carrier protein 2 and a 3-oxoacyl-CoA thiolase known as SCP2-thiolase (type-1)showed predominant expression in the midgut, and its coding SCP2 was involved in theabsorption and transport of cholesterol (Guo et al., 2009). In the silkworm, SCPx genehas been cloned (Gong et al., 2006). It presented expressions in the midgut, fat body, andhead on day 3 of the fifth-instar larvae in the silkworm (Fig. 6B), which suggested that theSCP2 protein might have a similar function with that of S. litura (Guo et al., 2009). Moreimportant, the SCP2-thiolase (type-1) encoded by SCPx plays a crucial role in the oxidationof the branched side chain of cholesterol to form bile acids in vertebrates (Ferdinandusseet al., 2000), while the physiological role has not been characterized in insects. Fortunately,the expression of the SCP2-thiolase (type-1) has also been detected in the prothoracicglands of Spodoptera littoralis, which are the main tissue producing the insect moltinghormone (Takeuchi et al., 2004). Thus, whether SCP2-thiolase (type-1) of the silkwormand other insects play role in the oxidation of cholesterol and participates in ecdysonesynthesis needs further study.

In insects, juvenile hormone (JH) is an important regulator for growth and development(Kinjoh et al., 2007) and several thiolases have been cloned and suggested to be related toJH biosynthesis (Kinjoh et al., 2007; Zhu et al., 2016; Zhang et al., 2017). Acetoacetyl-CoAthiolase catalyzes two molecules of acetyl-CoA to form acetoacetyl-CoA, which is thefirst enzyme in JH biosynthesis (Kinjoh et al., 2007; Zhang et al., 2017). The candidateacetoacetyl-CoA thiolases related to JH biosynthesis were cloned in B. mori andHelicoverpaarmigera (Kinjoh et al., 2007; Zhang et al., 2017). In this study, those two acetoacetyl-CoAthiolase genes were classified as T2-thiolases (BmorT2 and HarmT2), and they shared highsequence identities with the other T2-thiolases (Table S4). For example, BmorT2-thiolaseshared 82.71% sequence identity with HarmT2. In H. armigera, temporal expressions ofHarmT2-thiolase keep pace with JH fluctuations, and its expression can be inhibited bya juvenile hormone analog (Zhang et al., 2017). The expression of BmorT2-thiolase wasrelatively abundant in the head where the JH synthetic gland, corpora allata (CA), is located(Figs. 6A and 6B). Interestingly, we found BmorTFE- and BmorT1-1-thiolase also showedhigh expressions in the larval head (Fig. 6A). In humans, TFE- and T1-thiolases catalyzethiolytic cleavage of 3-ketoacyl-CoA into acetyl-CoA and acyl-CoA (Anbazhagan et al.,2014; Xia et al., 2019). However, T1-thiolase has been found synthetic and degradativeactivities in Ostrinia scapulalis (Lepidoptera: Crambidae). Therefore, whether T2-, TFE-and T1-thiolases were involved in JH biosynthesis is still worthy of experimental validation.

Acetyl-CoA is often used as the initial precursor for sex pheromone biosynthesis in insects(Matsumoto, 2010). Degradative thiolases may supplement with sufficient acetyl-CoA forsex pheromone synthesis (Brabcova et al., 2015). In this study, expression profiles of thethiolase genes were detected in the sex pheromone glands at different developmentalstages in the silkworm (Fig. 6C). Relatively, BmorSCP2 (type-1) maintains a high level of

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expression in the PGs on day 4 of pupae to 24-h-old virgin female moth. However, itsexpression level was sharply declined in themated female PGs (Fig. 6C). The previous studysuggested that an over 6-h mating duration can terminate the sex pheromone productionin the silkworm (Ando et al., 1996). The expression pattern of BmorSCP2 (type-1) wasconsistent with sex pheromone production (Matsumoto, 2010), which suggested that itmight be involved in sex pheromone biosynthesis. Generally, it is tempting to assume thata thiolase expressed in a specific tissue might obtain a specific role. Thus, the functionaldiversification and physiological roles of insect thiolases need yet further experimentalvalidation.

CONCLUSIONSIn the present study, genome-wide identification of the thiolase gene family was conductedin multiple insect genomes. A total of 137 thiolase genes were identified in 20 insects fromseven orders. About 80% of the thiolase genes have two ormore introns, and its exon/intronstructures reserve diversification. Based on the prediction, all the thiolase proteins arelocated in the mitochondria, cytosol, or peroxisome, and thiolases of the same class oftenhave similar cellular localization. Four highly conserved sequence fingerprints were foundin the insect thiolase proteins, including CxS-, NEAF-, GHP-, and CxGGGxG-motifs.Homology modeling analysis indicated that 3D structures of the insect thiolases sharesimilar to mammals, fishes, and microorganisms. Expression pattern analysis suggestedsome thiolase genes may be involved in steroid metabolism, JH, and sex pheromonebiosynthesis pathways in B. mori. These results might provide valuable information for thefunctional exploration of thiolase proteins in insects.

ACKNOWLEDGEMENTSThe author sincerely thanks the anonymous reviewers for their affirmation and constructivecomments on the manuscript.

ADDITIONAL INFORMATION AND DECLARATIONS

FundingThis study was supported by the Initiation Fund (No. 15E022) and the Teaching ReformResearch Project (No. Jgxmyb18151) of China West Normal University. The funders hadno role in study design, data collection and analysis, decision to publish, or preparation ofthe manuscript.

Grant DisclosuresThe following grant information was disclosed by the author:Initiation Fund: 15E022.China West Normal University: Jgxmyb18151.

Competing InterestsThe authors declare there are no competing interests.

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Author Contributions• Shou-Min Fang conceived and designed the experiments, performed the experiments,analyzed the data, prepared figures and/or tables, authored or reviewed drafts of thepaper, and approved the final draft.

Data AvailabilityThe following information was supplied regarding data availability:

The raw data and the uncropped blots are available as Supplemental Files.

Supplemental InformationSupplemental information for this article can be found online at http://dx.doi.org/10.7717/peerj.10393#supplemental-information.

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