The basic helix–loop–helix transcription factors in the Colorado potato beetle Leptinotarsa decemlineata

    The basic helix-loop-helix transcription factors in the Colorado potato beetle Leptinotarsa decemlineata Kai-Yun Fu, Qing-Wei Meng, Feng-Gong L¨u, Wen-Chao Guo, Tursun Ahmat, Guo-Qing Li PII: DOI: Reference:

S1226-8615(15)00021-7 doi: 10.1016/j.aspen.2015.01.007 ASPEN 618

To appear in:

Journal of Asia-Pacific Entomology

Received date: Revised date: Accepted date:

15 December 2014 20 January 2015 24 January 2015

Please cite this article as: Fu, Kai-Yun, Meng, Qing-Wei, L¨ u, Feng-Gong, Guo, WenChao, Ahmat, Tursun, Li, Guo-Qing, The basic helix-loop-helix transcription factors in the Colorado potato beetle Leptinotarsa decemlineata, Journal of Asia-Pacific Entomology (2015), doi: 10.1016/j.aspen.2015.01.007

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ACCEPTED MANUSCRIPT The basic helix-loop-helix transcription factors in the Colorado

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potato beetle Leptinotarsa decemlineata

Kai-Yun Fu1†, Qing-Wei Meng1†, Feng-Gong Lü1, Wen-Chao Guo2, Tursun Ahmat2, Guo-Qing Li1*

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1. Education Ministry Key Laboratory of Integrated Management of Crop Diseases

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and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China

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Urumqi 830091 , China

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2. Department of Plant Protection, Xinjiang Academy of Agricultural Sciences,

Running Head: bHLH transcription factors in Leptinotarsa

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Email address for all authors: Kai-Yun Fu, [email protected] Qing-Wei Meng, [email protected] Feng-Gong Lü, [email protected] Wen-Chao Guo, [email protected] Tursun Ahmat, [email protected] *Correspondence

to

Guo-Qing

Li,

+86-25-84395248 † Co-first author 1

Email:

[email protected].

Tel/Fax:

ACCEPTED MANUSCRIPT Abstract The basic helix-loop-helix (bHLH) transcription factors possess crucial functions

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in cell proliferation, determination, differentiation, cell cycle maintenance, and

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homeostasis or stress response pathways. Moreover, some bHLH proteins can activate gene transcription in response to environmental toxins. Since a few juvenile hormone analogues such as hydroprene, methoprene and pyriproxifen targeting a bHLH

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member Met are already registered for pest management, there is a potential to

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develop more insecticides targeting various bHLHs. Identification of bHLH members is the first step. Based on the transcriptome and the genome of the Colorado potato

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beetle Leptinotarsa decemlineata, the most important pest in potato, 49 bHLH

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members were identified. All LdbHLH members were defined by their names and

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families according to various phylogenetic analyses with bHLH homologues of D. melanogaster, A. mellifera, B. mori and T. castaneum. Among these LdbHLHs, 17, 10,

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10, 1, 10 and 1 members belonged to A, B, C, D, E and F high-order groups, respectively. The results would provide useful background information for future studies on functions of bHLH proteins in the regulation of L. decemlineata development. Moreover, the bHLHs could serve as potential insecticide targets. These proteins could be used in small molecule screen, or in the development of RNAi-based pest management methods.

Key words: bHLH, identification, Leptinotarsa decemlineata, Genome

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ACCEPTED MANUSCRIPT 1. Introduction The Colorado potato beetle Leptinotarsa decemlineata (Say) is the most important

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pest in potato. Control of the beetle has mainly relied on the use of insecticides (Jiang

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et al., 2011; Jiang et al., 2010). This inevitably leads to the development of resistance to various insecticides in most major potato-growing areas of the world (Alyokhin, 2009; Alyokhin et al., 2008; Jiang et al., 2011; Jiang et al., 2012; Shi et al., 2012).

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There is an urgent need for development of insecticides working through new target

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sites. Since a few juvenile hormone (JH) analogues such as hydroprene, methoprene and pyriproxifen are already registered for pest management targeting putative JH

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receptor (methoprene resistance, Met), a basic helix-loop-helix (bHLH) transcription

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factor (Bitra and Palli, 2013), there is a potential to develop more insecticides

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targeting various bHLH members.

The bHLH proteins comprise a large superfamily of transcription factors that

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possess crucial functions in cell proliferation, determination, differentiation, cell cycle maintenance, and homeostasis or stress response pathways (Hassan and Bellen, 2000; Jan and Jan, 1993; Massari and Murre, 2000; Murre et al., 1989; Weintraub, 1993). Moreover, some bHLH members decide cell fate in the developing central nervous system of embryo (Fisher and Caudy, 1998; Kageyama and Nakanishi, 1997). Structurally, bHLH proteins are typified by their highly conserved bHLH domain (Kadesh, 1993; Murre et al., 1989). The bHLH domain is about 60 amino acids long and comprises an upstream stretch of basic amino acid residues (b) and two helices separated by an intervening loop region of varying length (HLH). There are 19 highly 3

ACCEPTED MANUSCRIPT conserved sites within the bHLH motif and that a protein with 11 of the 19 conserved amino acids is most likely a bHLH protein (Atchley et al., 1999). The HLH domain

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promotes dimerization, resulting in the formation of homodimeric or heterodimeric

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complexes between bHLH members. The two basic domains that are brought together through dimerization are involved in recognition of and binding to specific hexanucleotide DNA sequences (Kadesh, 1993; Murre et al., 1989). However, the

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sequences flanking the bHLH domain are either nonhomologous or are too divergent

Morgenstern and Atchley, 1999).

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to yield a meaningful alignment (Atchley and Fitch, 1997; Atchley et al., 1999;

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The establishment of this predictive motif led to a quick expansion of identified

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bHLH members in various organisms (Ledent et al., 2002; Li et al., 2006a; Li et al.,

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2006b; Liu and Zhao, 2010; Zheng et al., 2009). In Insecta, 59, 55, 55 and 57 bHLH members have been found in Dipteran species Drosophila melanogaster (Simionato et

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al., 2007), Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus, respectively (Zhang et al., 2013); and 51 and 57 bHLH sequences have been documented in Hymenopteran species Apis mellifera (Wang et al., 2008) and Harpegnathos saltator (Liu et al., 2012), respectively. Moreover, 52, 51 and 54 bHLH proteins have been reported in Lepidopteran Bombyx mori (Huang et al., 2012; Wang et al., 2007), Coleopteran Tribolium castaneum (Bitra and Palli, 2010) and Hemipteran Acyrthosiphon pisum (Dang et al., 2011a), respectively. Currently, bHLH proteins are classified into A, B, C, D, E and F high-order groups (Ledent and Vervoort, 2001; Simionato et al., 2007). 4

ACCEPTED MANUSCRIPT To obtain potential bHLH members in L. decemlineata in the present study, we used amino acid sequences of 59 D. melanogaster bHLH motifs to search (Atchley

Shi

et

al.,

2013)

and

the

genome

data

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2014;

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and Fitch, 1997) against the transcriptome (Kontogiannatos et al., 2013; Kumar et al.,

(https://www.hgsc.bcm.edu/arthropods/colorado-potato-beetle-genome-project). Subsequent examination and analyses led us to successfully identify and define 49

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bHLH members in L. decemlineata. These results provide potential insecticide targets

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and useful information for future studies on structures and functions of bHLH proteins in L. decemlineata.

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2.1. Insect rearing

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2. Materials and methods

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Post-diapause L. decemlineata adults were collected from potato fields in each spring at Urumqi city (43.82N, 87.61E), Xinjiang Uygur autonomous region in China.

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Insects were routinely reared in an insectary according to a previously described method (Shi et al., 2013), using potato foliage at vegetative growth or tuber initiation stages.

2.2. Homology searches Amino acid sequences of 59 bHLH members in D. melanogaster (DmbHLH) and 51 bHLHs in T. castaneum (TcbHLH) were obtained from the online supplementary material of previous reports (Simionato et al., 2007). Each DmbHLH and TcbHLH sequence was used to search against the transcriptome (Kontogiannatos et al., 2013; Kumar

et

al.,

2014;

Shi

et

al., 5

2013),

and

genome

data

ACCEPTED MANUSCRIPT (https://www.hgsc.bcm.edu/arthropods/colorado-potato-beetle-genome-project).

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the retrieved expressed sequence tags (ESTs) were used as queries to undertake

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similarity searches using blastx algorithm against the NCBI protein database to

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ascertain that they contained or corresponded to genuine bHLH domains. 2.3. Total RNA isolation, cDNA synthesis and PCR

Total RNA was isolated from a mixed sample including eggs, the first-, second-,

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third-, and fourth-instar larvae, pupae and sexually mature adults following the

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standard protocol of TRIzol reagent (Invitrogen, Shanghai., China). First-strand cDNA of the sample was synthesized from 1 µg of total RNA template using Moloney

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Oligo (dT) 18 primer.

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Murine Leukemia Virus reverse transcriptase (Takara Bio, Dalian, China) and an

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Polymerase chain reaction (PCR) was performed to substantiate the selected bHLH ESTs. The primers based on the sequences were designed using Premier

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Primer 5.0 (Premier Biosoft Interpairs, Palo Alto, CA). Once initial bHLH sequences were authenticated, they were aligned to the full sequences of their corresponding members from D. melanogaster and T. castaneum. Some short sequence gaps between two aligned unigenes were found. Specific primers were designed based on the two sequences between each gap, and the gap was filled by RT-PCR. The final cDNA sequences were verified using the primers listed in Table 1. 2.4. Phylogenetic analysis To define orthology for each identified L. decemlineata bHLH member, phylogenetic analyses were carried out in two steps. Firstly, the 49 full LdbHLH 6

ACCEPTED MANUSCRIPT proteins were searched against Conserved Domain Database in NCBI and LdbHLH motifs were aligned using ClustalW and a phylogenetic tree was constructed by the

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maximum-likelihood (ML) (Guindon et al., 2010) method selecting the best-fitting

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model of amino acid (LG+G) substitution after estimation by ProtTest v3.3 (Darriba et al., 2011). The reliability of ML tree topology was evaluated by bootstrapping a sample of 1,000 replicates. Minimum evolution ME and neighbor joining NJ distant

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trees were conducted by using MEGA6 (http://www.megasoftware.net/) based on a

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poisoon correction model. Secondly, each LdbHLH motif sequence was used to conduct within-group phylogenetic analyses with D. melanogaster and T. castaneum

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bHLH motif sequences; that is, the amino acid sequence of each beetle bHLH motif

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was used to construct NJ phylogenetic trees with D. melanogaster and T. castaneum

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bHLH family members of the corresponding high-order group. The NJ trees were bootstrapped with 1000 replicates to provide information about their statistical

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reliability.

3. Results and discussion

3.1. bHLH members

In total, 49 putative bHLH members were identified based on the L. decemlineata transcriptome (Kontogiannatos et al., 2013; Kumar et al., 2014; Shi et al., 2013) and the genome. These novel bHLHs were submitted to GenBank database (GenBank accession

No. KP147911-KP147947,

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KP641319-KP641330).

Since

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decemlineata possesses less bHLH members than Dipteran species D. melanogaster 7

ACCEPTED MANUSCRIPT (Simionato et al., 2007), A. aegypti, A. gambiae and C. quinquefasciatus, respectively (Zhang et al., 2013); Hymenopteran species H. saltator (Liu et al., 2012) and A.

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mellifera (Wang et al., 2008); Lepidopteran B. mori (Huang et al., 2012; Wang et al.,

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2007); Coleopteran T. castaneum (Bitra and Palli, 2010) and Hemipteran A. pisum (Dang et al., 2011a), we may not find all bHLH members in L. decemlineata.

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Currently, bHLH proteins are classified into six high-order groups, namely A, B, C, D, E and F (Ledent and Vervoort, 2001; Simionato et al., 2007). Examination of the

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19 conserved amino acid sites and sequence alignment revealed that these 49 bHLH

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members belonged to 6 groups. There were 17, 10, 10, 1, 10 and 1 LdbHLH members

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in groups A, B, C, D, E and F, respectively (Figure 1).

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We adopted nomenclature used in D. melanogaster (Simionato et al., 2007) and T. castaneum (Bitra and Palli, 2010) for facilitating further studies on structural and

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functional comparison. In case that one D. melanogaster or T. castaneum bHLH sequence has two or more L. decemlineata homologues, we use 1, 2 and 3, or a, b and c etc. to number them. For instance, 5 homologues of the D. melanogaster HES genes were found in L. decemlineata. Therefore, these LdbHLH genes were named LdHES1a, LdHES1b, LdHES1c, LdHES2a and LdHES2b, respectively. Names of 49 LdbHLHs in accordance with their corresponding D. melanogaster and T. castaneum homologues are listed in Figure 1 and Table 2.

All LdbHLH sequence alignments have a gap in the basic region created by the amino acids DPMSHR of LdStich1. In contrast, such a gap did not find in all known 8

ACCEPTED MANUSCRIPT insect bHLH members in D. melanogaster (Simionato et al., 2007), A. aegypti, A. gambiae, C. quinquefasciatus (Zhang et al., 2013), H. saltator (Liu et al., 2012), A.

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mellifera (Wang et al., 2008), B. mori (Huang et al., 2012; Wang et al., 2007) and A.

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pisum (Dang et al., 2011a). Moreover, a gap created by the amino acids NSKTTL of LdHES2b has been found in the loop in alignments of LdbHLHs. Similar gaps have been found in the insect bHLH members in D. melanogaster (Simionato et al., 2007),

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A. aegypti, A. gambiae, C. quinquefasciatus (Zhang et al., 2013), H. saltator (Liu et

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al., 2012), A. mellifera (Wang et al., 2008), B. mori (Huang et al., 2012; Wang et al., 2007) and A. pisum (Dang et al., 2011a) (Figure 1).

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3.2 Identification of orthologous genes

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To inspect the evolutionary relationships of L. decemlineata bHLHs, molecular phylogenetic analyses were performed using a putative full set of bHLHs from L.

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decemlineata, D. melanogaster (Simionato et al., 2007) and T. castaneum (Bitra and Palli, 2010). Phylogenetic trees were constructed by NJ, ML, and NE (Table 2, Figure 2).

Group A bHLH proteins mainly regulate neurogenesis, myogenesis and mesoderm formation (Dang et al., 1992; Murre et al., 1989; Van Doren et al., 1991). Proteins in this group are inclined to bind core hexanucleotide DNA sequences referred to as E box (CACCTG or CAGCTG) (Ledent and Vervoort, 2001). This group contains 48-related-1/Fer1, 48-related-2/Fer2, ASCa, ASCb, ASCc, amber, 2 Atonal, Beta3, Delilah, E12/E47, Hand, mesp, Mist, MyoD, MyoRa, MyoRb, Net, NeuroD, 9

ACCEPTED MANUSCRIPT Neurogenin, NSCL, Oligo, paraxis, peridot, PTFa/Fer3, SCL, and Twist families (Simionato et al., 2008; Simionato et al., 2007). Candidates of NSCL, Atoanl, Beta3,

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PTFa, PTFb, Twist, ASCa, Delilah, E12/E47, Hand, Mist, MyoD, MyoRa, Net,

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Paraxis and SCL were identified in L. decemlineata. They were named as LdNSCL, LdAto, LdAmos, LdOli, LdFer1, LdFer3, LdTwi, LdAse2, LdDel, LdDa, LdHand, LdMistr, LdNau, LdMyoRa, LdNet, LdPxs and LdSCL, respectively (Figures 1 and 2,

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Tables 2 and 3). The bHLH proteins in NSCL, 2 Atoanl, Beta3, PTFa, PTFb, Twist,

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E12/E47, Hand, MyoD, MyoRa, Net, Paraxis and SCL families presented a high level of 1:1 orthology with those from other insect genomes, suggesting functional

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conservation of these bHLHs (Tables 2 and 3).

Group B members are mainly involved in cell proliferation and differentiation,

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sterol metabolism and adipocyte formation, and expression of glucose-responsive genes (Dang et al., 1992; Murre et al., 1989; Van Doren et al., 1991). Group B

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recognizes and binds E box sequence CACGTG or CATGTTG (Ledent and Vervoort, 2001). This group has AP4, Fig-α, MAD, max, MITF, MLX, Mnt, Myc, SREBP, TF4, and USF families (Simionato et al., 2007). A member belonging to AP4, MAD, Max, Mitf, MLX, Myc, SREBP, TF4 and USF families was found in L. decemlineata, and was designated LdCrp, LdMad, LdMax, LdMITF, LdMLX, LdMnt, LdDm, LdSREBP, LdBmx and LdUSF respectively (Figures 1 and 2, Tables 2 and 3). A high level of 1:1 orthology of these bHLH families with those from other insect genomes suggests functional conservation (Tables 2 and 3).

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ACCEPTED MANUSCRIPT C group proteins are responsible for the regulation of midline and tracheal development, circadian rhythms, and for the activation of gene transcription in

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response to environmental toxins (Ledent and Vervoort, 2001). Proteins in this group

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tend to bind DNA core sequence of ACGTG or GCGTG (Ledent and Vervoort, 2001). C group proteins contain one or two PAS domains following the bHLH motif (Ledent and Vervoort, 2001). The PAS domain is named after three proteins, D. melanogaster

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period (Per), the human aryl hydrocarbon receptor nuclear translocator (ARNT) and D.

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melanogaster single-minded (Sim) (Zelzer et al., 1997). Candidates belonging to AHR, SRC, ARNT, Bmal, HIF, Sim and Trh families were discovered in L.

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decemlineata, and designated LdSs, LdDys, LdTai, LdTgo, LdCyc, LdSima, LdSim and

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LdTrh respectively. Clock family has two members in L. decemlineata. They were

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named as LdClk and LdRst(1)JH (Figures 1 and 2, Tables 2 and 3). LdClk is a core circadian clock gene, whereas LdRst(1)JH encodes Met protein which mediates JH

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signaling pathway (Jindra et al., 2013).

Group D proteins correspond to bHLH proteins that are unable to bind DNA due to lack of a basic domain (Ledent and Vervoort, 2001). This group includes Extramacrochaete (Emc) protein (Ellis et al., 1990; Garrell and Modolell, 1990), which act as antagonists of Group A bHLH proteins (Van Doren et al., 1991; Van Doren et al., 1992). One candidate gene, LdEmc, was identified in the present survey (Figures 1 and 2, Tables 2 and 3).

Group E proteins are mainly involved in embryonic segmentation, somitogenesis 11

ACCEPTED MANUSCRIPT and organogenesis (Ledent and Vervoort, 2001). Proteins in this group bind preferentially to sequences referred to as N box (CACGCG or CACGAG) (Ledent

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and Vervoort, 2001). This group contains H/E(spl), Hey, and clockwork orange

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families (Gyoja and Satoh, 2013; Simionato et al., 2007). Many members of this high-order group have a hairy/orange domain. H/E(spl) and Hey proteins often have a WRPW and a YRPW motif at its C-terminus, respectively (Gyoja et al., 2012). In L.

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decemlineata, eight H/E(spl) members were found, and named as LdDpn, LdH,

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LdHES1a, LdHES1b, LdHES1c, LdHES2a and LdHES2b, and LdSide respectively. Obviously, HES proteins showed a species-specific bloom in L. decemlineata. Five

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members (LdHES1a, LdHES1b, LdHES1c, LdHES2a and LdHES2b) were identified.

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Moreover, two Hey members were identified, and named as LdHey and LdStich1,

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respectively (Figures 1 and 2, Tables 2 and 3).

Group F proteins mainly regulate head development and formation of olfactory

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sensory neurons (Dang et al., 2011b; Ledent and Vervoort, 2001). Only a single orthologous family, COE, is categorized in group F (Simionato et al., 2007). One candidate gene (LdKn) of this high-order group was identified in the present paper (Figures 1 and 2, Tables 2 and 3).

3.3 A comparison of insect bHLH members

The available bHLH members from A. mellifera (Wang et al., 2008), B. mori (Huang et al., 2012; Wang et al., 2007), T. castaneum (Bitra and Palli, 2010), D. melanogaster (Simionato et al., 2007) and L. decemlineata are listed in Table 3. It is 12

ACCEPTED MANUSCRIPT found that all these five insects have no members in ASCb, MyoRb, Oligo, and Figα (Table 3). Moreover, these members are not found in the genomes of H. saltator (Liu

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et al., 2012), A. aegypti, A. gambiae and C. quinquefasciatus, respectively (Zhang et

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al., 2013). It is suggested that ASCb, MyoRb, Oligo, and Figα members may have no role in insect species, or their physiological functions have been replaced by other

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transcription factors.

We did not find NeuroD in A. mellifera, B. mori, D. melanogaster, L. decemlineata

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(Table 3), A. gambiae, C. quinquefasciatus (Zhang et al., 2013) and A. pisum genomes

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(Dang et al., 2011a). However, the bHLH was present in T. castaneum (Table 3) and

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H. saltator genomes (Liu et al., 2012). Moreover, Mad is present in both L. decemlineata transcriptome and T. castaneum genome (Table 3), as well as in A.

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pisum genome (Dang et al., 2011a). However, it is absent in A. mellifera, B. mori, D. melanogaster (Table 3), H. saltator (Liu et al., 2012), A. aegypti, A. gambiae and C.

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quinquefasciatus genomes (Zhang et al., 2013). The data suggest that these bHLHs may play different roles in the adaptation of the different insect species to their specific biological niches.

Conclusion Our study identified 49 bHLH members in L. decemlineata. All LdbHLH members have been defined by their names and families according to various phylogenetic analyses with bHLH homologues of D. melanogaster, A. mellifera, B. mori and T. castaneum. Our results facilitate the development of potential insecticides 13

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Acknowledgments

This research was supported by the National Natural Sciences Foundation of China

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(31272047 and 31360442), a nationally special fund of China for agri-scientific research in the public interest (201103026) and a nationally special fund of China for

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agri-scientific research in the public interest (201103026).

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basic/helix-loop-helix transcription factor family in rice and Arabidopsis. Plant physiology 141, 1167-1184. Liu, A., Wang, Y., Dang, C., Zhang, D., Song, H., Yao, Q., Chen, K., 2012. A genome-wide identification and analysis of the basic helix-loop-helix transcription factors in the ponerine ant, Harpegnathos saltator. BMC Evolutionary Biology 12, 165. Liu, N., Pan, L., Miao, J., Xu, C., Zhang, L., 2010. Molecular cloning and sequence analysis and the response of an aryl hydrocarbon receptor homologue gene in the clam Ruditapes philippinarum exposed to benzo(a)pyrene. Comparative Biochemistry and Physiology C: Toxicology and Phamacology 152, 279-287. Liu, W.Y., Zhao, C.J., 2010. Genome-wide identification and analysis of the chicken basic helix-loop-helix factors. Comparative and Functional Genomics 2010, 682095. Massari, M.E., Murre, C., 2000. Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms. Molecular and Cellular Biology 20, 429-440. Misra, J.R., Horner, M.A., Lam, G., Thummel, C.S., 2011. Transcriptional regulation of xenobiotic detoxification in Drosophila. Genes and Development 25, 1796-1806. Morgenstern, B., Atchley, W.R., 1999. Evolution of bHLH transcription factors: modular evolution by domain shuffling? Molecular Biology and Evolution 16, 1654-1663. Murre, C., McCaw, P.S., Vaessin, H., Caudy, M., Jan, L.Y., Jan, Y.N., Cabrera, C.V., Buskin, J.N., Hauschka, S.D., Lassar, A.B., Weintraub, H., Baltimore, D., 1989. Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell 58, 537-544. Rowlands, J.C., Gustafsson, J.A., 1997. Aryl hydrocarbon receptor-mediated signal transduction. Critical Review of Toxicology 27, 109-134. Schecter, A., Birnbaum, L., Ryan, J.J., Constable, J.D., 2006. Dioxins: an overview. Environmental Research 101, 419-428. Shi, X.-Q., Guo, W.-C., Wan, P.-J., Zhou, L.-T., Ren, X.-L., Tursun, A., Fu, K.-Y., Li, G.-Q., 2013. Validation of reference genes for expression analysis by quantitative real-time PCR in Leptinotarsa decemlineata (Say). BMC Research Notes 6, 93. Shi, X.-Q., Xiong, M.-H., Jiang, W.-H., Wang, Z.-T., Guo, W.-C., Xia, Z.-H., Fu, W.-J., Li, G.-Q., 2012. Efficacy of endosulfan and fipronil and joint toxic action of endosulfan mixtures against Leptinotarsa decemlineata (Say). Journal of Pest Science 85, 519-526. Simionato, E., Kerner, P., Dray, N., Le Gouar, M., Ledent, V., Arendt, D., Vervoort, M., 2008. Atonal- and achaete-scute-related genes in the annelid Platynereis dumerilii: insights into the evolution of neural basic-helix-loop-helix genes. BMC Evolutionary Biology 8, 170. Simionato, E., Ledent, V., Richards, G., Thomas-Chollier, M., Kerner, P., Coornaert, D., Degnan, B.M., Vervoort, M., 2007. Origin and diversification of the basic helix-loop-helix gene family in metazoans: insights from comparative genomics. BMC Evolutionary Biology 7, 33. 17

ACCEPTED MANUSCRIPT Doren, M., Ellis, H.M., Posakony, J.W., 1991. The Drosophila extramacrochaetae protein antagonizes sequence-specific DNA binding by daughterless/achaete-scute protein complexes. Development 113, 245-255 Van Doren, M., Powell, P.A., Pasternak, D., Singson, A., Posakony, J.W., 1992. Spatial regulation of proneural gene activity: auto- and cross-activation of achaete is antagonised by extramacrochaete. Genes and Development 6, 2592-2605 Wang, Y., Chen, K., Yao, Q., Wang, W., Zhu, Z., 2007. The basic helix-loop-helix transcription factor family in Bombyx mori. Development Genes and Evolution 217, 715-723. Wang, Y., Chen, K., Yao, Q., Wang, W., Zhu, Z., 2008. The basic helix-loop-helix transcription factor family in the honey bee, Apis mellifera. Journal of Insect Science 8, 40. Weintraub, H., 1993. The MyoD family and myogenesis: redundancy, networks, and thresholds. Cell 75, 1241-1244. Zelzer, E., Wappner, P., Shilo, B.Z., 1997. The PAS domain confers target gene specificity of Drosophila bHLH/PAS proteins. Genes and Development 11, 2079-2089 Zhang, D.B., Wang, Y., Liu, A.K., Wang, X.H., Dang, C.W., Yao, Q., Chen, K.P., 2013. Phylogenetic analyses of vector mosquito basic helix-loop-helix transcription factors. Insect Molecular Biology 22, 608-621. Zhao, X., Salgado, V.L., 2010. The role of GABA and glutamate receptors in susceptibility and resistance to chloride channel blocker insecticides. Pesticide Biochemistry and Physiology 97, 153-160. Zheng, X., Wang, Y., Yao, Q., Yang, Z., Chen, K., 2009. A genome-wide survey on basic helix-loop-helix transcription factors in rat and mouse. Mammlian Genome 20, 236-246.

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ACCEPTED MANUSCRIPT Table 1 Primers used in RT-PCR for 49 novel bHLH cDNAs in L. decemlineata Reverse primer sequences (5′ - 3′ )

KP147922 KP147928 KP147930 KP147932 KP147933 KP147934 KP147940 KP147942 KP147945 KP147947 KP641319 KP641327 KP641325 KP641328 KP641326 KP641324 KP641320

CTGTAGTGTTGTTTTTTCGGCG TACATAAGTCAGTCGGTCGCA ATTGTGGTGGGTTTCAGTTAGTG ATAATTCTTGCCTAAGACGGAGT TTCAGTACTTCAATATTACGCGC CAAAATGGCTACCAGTGATGATG CGACAGTGACGATGAGAGG GGGACGTTTACTACGACATCA CGAGGGAGAGAGATAGAACACAC AAGAGTCGTAAAAAGGAGGTCAA AGAGGATAAGGGTGGAAG CCGTCAAATACACCAGAA GTCCCTGATGGAATCTTT ACCACTCGGCAGAATCAA AAACGGCAACGGGAATAG TCTTACCGTTACAGTGCC TCATAGTGCCTTCAACCC

ACCTTGAAAACACTTAGAAAATA TAGATTCAAAACCTAGGAACTCG AAAAACCGCATAGTGATTGGA TTGGTCAAAAGGGGATAAGC GCACTTAACGCAATAACGACA ATGTCTAAACAGTTCCGCAAATA TTGAGAAAGAATCGCTATTGAACT ACCACTGCCCACTTGTCA ATGCCCTGCTATGAGCTGCGTT CAATTTCCCATATTTTATCCACTC TCTGGAGGAAGAGTAGGC TCACATAGAACCTGACCCA TGCCATCTGTAGGGTCTC TGTGCCATCTGCAAAGTC AAAGCACTGCACGGAATT AGTTTCTATACGGGAAAGTC TATTCCAGTCCCCTTCCA

KP147915 KP147916 KP147917 KP147920 KP147924 KP147925 KP147926 KP147927 KP147929 KP147931

CACAGCGTCAGTCTTCAGCC TCCCGTAGAAAGCGTTGC CCATCGCATCACATGACCT CATGGACGAAGACCTTGCTAAT TCCGTTGCGAGTTGCGAC GATCGTAGTTGTATGTTTTTGTC GGAAGCCAGTATTATTCACAGG GCGATTGTCGTCGGTGG GTCCTTGATCTTTCACTGGCAG GTTCGATAAGTGGAGATTATGTCTT

CCGTGGATATTTTCGTCAGC GTCTGACGCTGAGTGATCCTAT CCTCCATTTCTTGCTCCTTTT AAACGCCATCCACCATT AAAATAACCATACGCCACCAAT TTGGCTGCATTAACCTTTA AACCACAAAAGAAACTCAGG TTTTGGGATTCTCCGTCAGT ATGAGCAAGATGTATTAGTCGGTA CGATCACGATGCCTGTTTCA

TCAGAGGCAGGGTCAAGGT CACAACGGGAACGACATC TATAGACGGTCGCATAAT GACTCGATAGCTCAGTACATTAA ATTTACCTGGTGATGTCGC CTCAGGGGGACTGGAAAT TTAATTTCGTGTTTTTTTCTGTTT TACCATCACTCGGCACTAAAG ACAGGGCGTTTTGCGTCA TTTTCTCAGGGGGACTTGCTA

TGATGCTGATGCGGACTG AAAGGACCTGGGAAAAGA TAACCTCTCGAAAGAATT ACGGCACTCTCGCATAGC CCATAATAATCGTATCATTGAAG TGACATTGGTAAGTATTCGCT CCTACCCAAGCCTCACCATT CAGTGCACATATTCGTTAGCC GCTTCAAAGGTAGCGGACTG TCTGCTCATTCAGGTCAAGGC

KP641321 KP641329 KP147911 KP147914 KP147912 KP147913 KP147921 KP147936 KP147944 KP147946

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Forward primer sequences (5′ - 3′ )

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Group A LdNau LdNet LdMyoRa LdDel LdAse2 LdDa LdSCL LdMistr LdPxs LdHand LdNSCL LdOli LdAto LdAmos LdFer1 LdFer3 LdTwi Group B LdDm LdMnt LdUSF LdSREBP LdMad LdBmx LdMax LdMITF LdMLX LdCrp Group C LdSs LdDys LdRst(1)JH LdTai LdClk LdTgo LdSima LdSim LdTrh LdCyc Group D

GenBank number

AC

Name

19

ACCEPTED MANUSCRIPT GATTTAGTGCGGTTGCCTTTT

CAAATGACCCATCGGAAGTT

KP147918 KP147919 KP147935 KP147937 KP147938 KP147939 KP641323 KP641322 KP147941 KP147943

ACGACGACAGAAGCGATAGG GAGTGCCTTTAACCAAACTTTAC AAGTGCCGACTTAGTGCCA TCCGTAAACAGTTGAAGGAACGT GCGGGGAGTCATTCAGAGTTTA TGGAGTTGACAGTGCGGC ATGATACACGCAGTGTTCA GGAAAATCAGGAAACCCC ATGTGCTGCTTCTCCCCGA ATGGAAAAAAGAAGGAGAGCAA

ACCAGAACCCTTGACTACGAG TTACCACGGTCTCCATACTGAT ATTTCCTGTCCCGAACTCAAC CCCCTGACCATCACAGCCT GTAGGGGTGCTCCAGGTTAGG CAAAAGGTAAATTCGTCCTCAAT CTGCCTACTATTGACTTGCTAT TTCGCAATTATCACCAACAC TGCCGCTGAAATCCAATG GGAAGCCTACTGGGAATGAGC

KP641330

ATGTGCCGAGTTCTATTG

AGAGCAGGGGAACCATAA

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KP147923

Table 2. A complete list of basic helix-loop-helix (bHLH) proteins found in the L. decemlineata transcriptome dataset Family Name

Bootstrap values

D

Gene name

Group

Gene ID

Homolog

KP147933

A A

KP147932 KP147934

57 97

57 97

51 84

TcDel TcDa

A A

KP147947 KP147942

99 99

99 100

82 98

TcHand TcMistr

MyoD MyoRa

A A

KP147922 KP147930

99 100

97 100

86 60

TcNau TcMyoRa

Net Paraxis

A A

KP147928 KP147945

98 100

98 100

97 78

TcNet TcPxs

LdSCL LdNSCL

SCL NSCL

A A

KP147940 KP641319

94 91

94 89

59 73

TcSCL TcNSCL

LdOli LdAto

Beta3 Atonal

A A

KP641327 KP641325

70 100

64 100

58 88

TcOli TcAto

LdAmos LdFer1

Atonal PTFa

A A

KP641328 KP641326

99 100

98 98

90 98

TcAmos1 TcFer1

LdFer3 LdTwi

PTFb Twist

A A

KP641324 KP641320

75 96

76 94

68 70

TcFer3 TcTwi

LdCrp LdMax

AP4 Max

B B

KP147931 KP147926

99 98

95 97

87 95

TcCrp TcMax

LdMITF LdMLX

Mitf MLX

B B

KP147927 KP147929

99 100

99 100

68 98

TcMitf DmMlx

LdMnt LdMad

Mnt Mad

B B

KP147916 KP147924

97 100

99 100

55 99

TcMnt TcMad

LdDm LdSREBP

Myc SREBP

B B

KP147915 KP147920

73 94

70 94

99 78

TcDm TcSREBP

LdBmx LdUSF

TF4 USF

B B

KP147925 KP147917

99 97

99 97

80 93

TcBmx TcUSF

ASCa

LdDel LdDa

Delilah E12/E47

LdHand LdMistr

Hand Mist

LdNau LdMyoRa LdNet LdPxs

CE P

LdAse2

TE A

ME 56

AC

LdEmc Group E LdH LdHES1a LdHey LdStich1 LdHES1c LdHES1b LdHES2a LdHES2b LdSide LdDpn Group F LdKn

20

NJ 63

ML 78

TcAse2

ACCEPTED MANUSCRIPT SRC

C

KP147914

99

99

65

TcTai

LdSs LdDys

AHR AHR

C C

KP641321 KP641329

99 99

99 95

90 88

TcSs TcDys

LdTgo LdCyc

ARNT Bmal

C C

KP147913 KP147946

100 53

99 59

95 51

TcTgo DmCyc

LdClk LdRst(1)JH

Clock Clock

C C

KP147912 KP147911

56 99

58 98

59 62

TcClk TcRst(1)JH

LdSima LdSim

HIF Sim

C C

KP147921 KP147936

86 97

85 98

82 95

TcSima DmSim

LdTrh LdEmc

Trh Emc

C D

KP147944 KP147923

98 85

98 87

66 100

TcTrh TcEmc

LdDpn LdH

H/E(spl) H/E(spl)

E E

KP147943 KP147918

62 63

56 64

78 82

TcDpn TcH

LdHES1a LdHES1b

H/E(spl) H/E(spl)

E E

KP147919 KP147939

80 n/m

76 n/m

67 n/m

TcHES1 n/m

LdHES1c LdHES2a

H/E(spl) H/E(spl)

E E

KP147938 KP641323

n/m 99

n/m 99

n/m 89

n/m TcHES2

LdHES2b LdSide

H/E(spl) H/E(spl)

E E

KP641322 KP147941

69 99

59 99

n/m 95

TcHES2 TcSide

LdHey LdStich1

Hey Hey

E E

KP147935 KP147937

99 99

99 100

95 99

TcHey TcSTICH

LdKn

COE

F

KP641330

100

100

95

TcKn

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LdTai

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LdbHLH genes are named according to their D. melanogaster homologues. Bootstrap values are obtained from in-group phylogenetic analyses with T. castaneum or D. melanogaster bHLH motif sequences using NJ, MP, and ML algorithms, respectively. n/m means that a LdbHLH does not form a monophyletic group with any other single bHLH motif sequence.

Group A

AC

Table 3. A comparison on bHLH family members from five insect species Family name

A.m.

B.m.

T.c.

D.m.

L.d.

ASCa ASCb

2 0

4 0

3 0

4 0

1 0

Atonal Beta3

3 1

1 1

3 1

3 1

2 1

Delilah E12/E47

0 1

1 1

2 1

1 1

1 1

Hand Mesp

1 1

1 1

1 0

1 1

1 0

Mist MyoD

2 1

1 1

1 1

1 1

1 1

MyoRa MyoRb

1 0

1 0

1 0

1 0

1 0

Net NeuroD

1 0

1 0

1 1

1 0

1 0

Ngn NSCL

1 1

1 1

1 1

1 1

0 1

Oligo

0

0

0

0

0

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ACCEPTED MANUSCRIPT

D

1

1

1

PTFa PTFb

1 1

1 1

1 2

1 2

1 1

SCL Twist

1 1

1 1

1 1

1 1

1 1

AP4 Figα

1 0

1 0

1 0

1 0

1 0

Mad Max

0 1

0 1

1 1

0 1

1 1

MITF MLX

1 1

1 1

1 0

1 1

1 1

Mnt Myc

1 1

1 1

1 1

1 1

1 1

SREBP TF4

1 1

1 1

1 1

1 1

1 1

USF SRC

2 1

1 1

1 1

1 1

1 1

AHR ARNT

2 1

3 1

2 1

2 1

2 1

Bmal Clock

1 2

2 3

1 2

1 3

1 2

HIF Sim

1 1

1 1

1 0

1 1

1 1

Trh Emc

1 1

1 1

1 1

1 1

1 1

6 2

5 2

5 2

11 1

8 2

1 51

1 52

1 51

1 59

1 49

H/E(spl) Hey

F

COE

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E

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1

D

C

1

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B

Paraxis

AC

The bHLHs are from A.m. (Apis mellifera) (Wang et al., 2008), B.m. (Bombyx mori) (Huang et al., 2012; Wang et al., 2007), T.c. (Tribolium castaneum) (Bitra and Palli, 2010), D.m. (Drosophila melanogaster) (Simionato et al., 2007) and L.d. (L. decemlineata).

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Figure 1. Multiple sequence alignment of 49 basic helix-loop-helix (bHLH) motifs from L. decemlineata. The boundaries of the basic, helix 1, loop and helix 2 regions within the bHLH domain following Ferre-D’Amare et al. (1993) (Ferre-D'Amare et al., 1993). Identical amino acid residues are shaded in black, conserved ones in dark grey and less conserved ones in light grey. Sites with highly conserved amino acid residues are marked with triangles at the bottom.

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Figure 2. Phylogenetic relationship of 49 LdbHLH members. Other insect bHLH proteins are from Drosophila melanogaster (Dm), Tribolium castaneum (Tc), Anopheles gambia (Ag), Aedes egypti (Ae), Bombyx mori (Bm) Culex quinquefasciatus (Cq), Dendroctonus ponderosne (Dpo) and Danaus plexippus (Dpl). Six neighbor-joining (NJ) trees show bHLH members from groups A, B, C, D, E and F.

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Graphical abstract

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ACCEPTED MANUSCRIPT Highlights  49 basic helix-loop-helix (bHLH) transcription factors were identified

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 17, 10, 10, 1, 10 and 1 members belonged to A, B, C, D, E and F groups

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 Our results facilitate further researches

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