Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525 DOI: 10.13102/sociobiology.v68i4.5443Sociobiology 68(4): e5443 (December, 2021) Introduction Trehalose is a non-reducing disaccharide with two glycosidically linked glucose units present in all forms of life except mammals, including plants, bacteria, fungi, yeast, Abstract Trehalose provides the main energy source for the physiological activities of insects, especially in adverse conditions. Trehalase is the only enzyme that hydrolyzes trehalose, therefore it is important to clarify the distribution and expression of trehalase under adverse conditions such as high temperatures and starvation. Here, we have cloned the trehalase genes and investigated their expression in different tissues, at multiple development stages, and with the treatments of high temperature and starvation in Bombus lantschouensis, which is considered to be one of the most commercially viable native species in China. The results suggest that the membrane-bound (BlTre-2) cDNA has an open reading frame of 1986 nucleotides, which encodes a protein of 662 amino acids, and two putative transmembrane domains. qRT-PCR analysis indicated that BlTre-2 was expressed in 10 tissues and at nine development stages, with the highest expression in general in 30-day-old workers, and in ovarian tissue in particular. The expression of BlTre-1 for 15-day-old workers which were exposed to a pre-treatment of 45°C increased over the first 5 h and subsequently decreased over time. In contrast the expression of BlTre-2 consistently decreased over time. The highest expression levels of BlTre-1 and BlTre-2 were observed the newly emerged adult workers when starved for 16-20 h. These results indicate that BlTre-2 may be part of the carbohydrate metabolism of the bumblebee, and that BlTre-1 is a key gene regulating energy metabolism and providing trehalose when exposed to a high temperature. Both BlTre-1 and BlTre-2 may balance trehalose and provide energy when B. lantschouensis is starved. Sociobiology An international journal on social insects Jiamin Qin1,2, Feng Liu3, Jie Wu1, Shaoyu He4, Muhammad Imran5, Wen Lou3, Hongmei Li-Byarlay6, Shudong Luo1 Article History Edited by Marco Antonio Costa, UESC, Brazil Received 24 May 2020 Initial acceptance 22 November 2020 Final acceptance 26 June 2021 Publication date 19 November 2021 Keywords Bombus lantschouensis, trehalase, sequence analysis, stress conditions, gene expression. Corresponding author Shudong Luo Key Laboratory for Insect-Pollinator Biology Institute of Apicultural Research, CAAS Beijing 100093, China. E-Mail: luoshudong@caas.cn nematodes, and insects (Elbein et al., 2003; Thompson, 2003; Argüelles, 2014; Nardelli et al., 2019). In these organisms, trehalose plays an important role in protecting proteins and cellular membranes during unfavorable environmental conditions such as heat, desiccation, dehydration, freezing, 1 - Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Key Laboratory of Pollinating Insect Biology, Ministry of Agriculture and Rural Affairs, Beijing, China 2 - Sericulture and Apiculture Research Institute, Yunnan Academy of Agricultural Sciences, Mengzi, China 3 - Apiculture Institute of Jiangxi Province, Nanchang, China 4 - Eastern Bee Research Institute, Yunnan Agricultural University, Kunming, China 5 - Department of Entomology, The University of Poonch Rawalakot, Azad Jammu and Kashmir, Pakistan 6 - Agricultural Research and Development Program, Department of Agriculture and Life Science, Central State University, Wilberforce, OH, United States RESEARCH ARTICLE - BEES Molecular Characterization and Gene Expression of Trehalase in the Bumblebee, Bombus lantschouensis (Hymenoptera: Apidae) mailto:luoshudong@caas.cn Jiamin Qin et al. – Molecular Characterization and Gene Expression of Trehalase Bombus lantschouensis2 hyperosmosis, anhydrobiosis, and oxidation (Wyatt, 1967; Crowe et al., 1992; Thompson, 2003; Mitsumasu et al., 2010). As a major blood sugar of insects, trehalose is present in the hemolymph of larvae, pupae, and adults (Alumot et al., 1969; Becker et al., 1996; Thompson, 2003). Trehalose is the main metabolic source for physiological activity, such as growth, development (Łopieńska-Biernat et al., 2018), energy metabolism (Thompson, 2003), anhydrobiosis (Mitsumasu et al., 2010), feeding behavior (Tang et al., 2014; Yasugi et al., 2017), chitin synthesis (Tang et al., 2012, 2016, 2017, 2018; Shen et al., 2017; Zhang et al., 2017), and diapause (Kamei et al., 2011; Yang et al., 2013; Wang et al., 2018). On the other hand, it plays key roles in adverse circumstances, such as starvation (Tang et al., 2014; Shukla et al., 2014; Liu et al., 2016). In insects, trehalase (α-glucoside-1-glucohydrolase, EC 3.2.1.28) is the only key enzyme that catalyzes the conversion of one molecule of trehalose to two molecules of glucose utilized through glycolysis (Clegg & Evans, 1961; Tatun et al., 2008). Two types of trehalase (soluble and membrane-bound) and corresponding genes (Tre-1 and Tre- 2) have been cloned in a variety of insects (Qin et al., 2015; Liu et al., 2016; Shukla et al., 2016; Zhao et al., 2016). Both forms contain signature motifs (PGGRFEFYYWDSY and QWDYPNAWPP) and a highly conserved glycine-rich region (GGGGEY). TRE-1 and TRE-2 break down the intracellular and extracellular trehalose, respectively (Shukla et al., 2014; Liu et al., 2016). The Tre-1 gene, which was first cloned from Tenebrio molitor in 1992, is mainly found in the digestive and circulatory systems (Takiguchi et al., 1992). Tre-2 contains a putative transmembrane domain (Takiguchi et al., 1992; Tang et al., 2008; Gu et al., 2009; Forcella et al., 2010) and is mostly identified in the muscle (Mitsumasu et al., 2005). Recent studies have indicated that only Tre-1 and Tre-2 are found in the majority of insects, such as Apolygus lucorum (Tan et al., 2014), Helicoverpa armigera (Ai et al., 2018), Spodoptera exigua (Tang et al., 2008), Omphisa fuscidentalis (Tatun et al., 2008), Drosophila melanogaster (Shukla et al., 2016), Cnaphalocrocis medinalis (Tian et al., 2016), and Bemisia tabaci (Wang et al., 2014). Multiple Tre- 1 have been cloned in insects, including Harmonia axyridis (Tang et al., 2014; Shi et al., 2016), Locusta migratoria (Liu et al., 2016), Nilaparvata lugens (Zhao et al., 2016), Tribolium castaneum (Tang et al., 2016), and Leptinotarsa decemlineata (Shi et al., 2016). In addition, trehalase gene is expressed in various tissues inducing the diapause of silkworm eggs and improving the cold resistance (Kamei et al., 2011; Tan et al., 2014). Generally, two types of trehalase, TRE-1 and TRE-2 are involved in various physiological functions such as long- distance flight metabolism, chitin synthesis during molting, energy metabolism, muscular movement, reproduction, and cold tolerance (Chen et al., 2010; Tang et al., 2012; Zhang et al., 2012; Shi et al., 2019). Bumblebees (Apidae, Bombus Latreille), important pollinators of many endangered alpine plants and agricultural crops, play a major role in maintaining biodiversity of ecosystems and agricultural production (Williams & Osborne, 2009; Gunnarsson & Federsel, 2014). Many species of bumblebees (such as Bombus terrestris, B. impatiens, B. occidentalis, and B. ignitus) have been used for commercial crop pollination (Velthuis & van Doorn, 2006). B. lantschouensis (Hymenoptera: Apidae), once mistaken for B. hypocrita, is widely distributed in the North China and is a native bumblebee species used in commercial applications – it is recognized in Chinese agriculture to be an excellent pollinator (An et al., 2014). There is not much information is available on the function, structure, tissue distribution, and expression patterns of the trehalase gene in B. lantschouensis even though trehalase has been studied in honeybees (Alumot et al., 1969; Brandt & Huber, 1979; Lee et al., 2007; Łopieńska-Biernat et al., 2018). Understanding the fundamental molecular characteristics and function of trehalase will enable us to improve the population and health of B. lantschouensis. In the present study, we cloned and characterized the Tre-2 gene from B. lantschouensis and measured patterns of expression in tissues at different chronological ages. In addition, we investigated the expression patterns of both Tre- 1 and Tre-2 in B. lantschouensis at 45°C and under starvation conditions. Materials and Methods Bumblebees Newly mated queens of the bumblebee B. lantschouensis were collected from the Hebei Province of China. These queens were reared under controlled climatic conditions at a temperature of 29 ± 0.5°C and 60±5% relative humidity in continuous darkness. Queens and their newly emerging workers were fed a 50% sucrose solution and pollen (from Brassica campestris L. and Prunus armenica L.). To ensure the adult bumblebees were the same age, all new workers were marked on the thorax with a white mark within 1 h. The workers used in this study were 15 days old but particular exceptions, which are noted later on. All the samples were taken from three colonies, and each colony was treated as an independent replicate. RNA Extraction, cDNA Synthesis, and PCR The total RNA was extracted from the sample with Trizol (Invitrogen, Carlsbad, CA, USA). First-strand cDNA synthesis was synthesized from 1 μg of RNA using a Transcriptor First Strand cDNA Synthesis Kit (Takara, Dalian, China), and the undiluted first-strand cDNA was used as the template for the polymerase chain reaction (PCR). Conserved District Fragment Amplification To obtain a conservative fragment of BlTre-2 from B. lantschouensis, a pair of degenerate primers, Tre-2-F and Tre-2-R (Table 1), were designed based on the conserved Sociobiology 68(4): e5443 (December, 2021) 3 amino acid sequences of the known Tre-2 in B. terrestris and B. impatiens (GenBank accession nos. XP_003393687 and XP_003490073). The PCR amplification reaction system contained 2 μL cDNA template, 1 μL of each primer (10 μmol/L) and 12.5 μL of the PCR mix, and finally was topped to 25 μL volume with nuclease-free water. PCR conditions were as follows: 94°C for 5 min, 35 amplification cycles of 94°C for 40 s, 50°C for 30 s, and 72°C for 1 min, and then an extension at 72°C for 7 min. The PCR products were electrophoresed for 10 min under 200 volts and then excised from agarose gels (1%) and purified with DNA Purification Kits (Version 2.0, Takara). Purified PCR products (4.2 μL) were ligated into 0.8 μL pMD-19T vectors using 5 μL Solution I at 16°C. The ligated constructs were transformed into Tran1-T1 competent cells and cultured for 1 h at 37°C. We selected an ampicillin-resistant clone and sub-cultured in 600 μL LB liquid medium to obtain the optimum amount of the expression vector, which was sequenced using M13- forward and M13-reverse. To complete the 5’ and 3’ ends, a Transcriptor First Strand cDNA Synthesis Kit (Takara) was used to synthesize 5’-RACE and 3’-RACE first-strand cDNA according to the manufacturer’s instruction. Specific primers 5’-OGSP and 5’- OP, 5’-IGSP, and 5’-IP for 5’-RACE, and 3’-OGSP and 3’-OP, 3’-IGSP, and 3’-IP for 3’-RACE (Table 1) were synthesized based on the cDNA sequence of the PCR fragment. The first PCR amplification reaction system contained 2.5 μL cDNA template, 1 μL of each primer (10 μmol/L) (5’-OGSP and 5’-OP/3’-OGSP and 3’-OP), 1 μL dNTP Mix, 2.5 μL 10×EX Buffer, 0.4 μL EX Taq HS, and was finally topped to 25 μL volume with nuclease-free water. PCR was performed under the following conditions: 94°C for 2 min, 35 amplification cycles of 94°C for 15 s, 60°C for 30 s, and 72°C for 40 s, and an extension at 72°C for 10 min. The products of the first PCR were diluted one-fold and then used as a template (2.5 μL) for the second PCR. The second PCR had the same reagent and content as the first PCR except for the primers (5’-IGSP and 5’-IP/3’-IGSP and 3’-IP) and was performed under the following conditions: 2 min at 94°C followed by 30 cycles of 15 s at 94°C, 30 s at 60°C, and 40 s at 72°C, and then 10 min at 72°C. Protein and cDNA Sequence Analyses The cDNA sequence of BlTre-2 was analyzed to determine its similarity with Tre-2 genes of B. terrestris (XP_003393687) and B. impatiens (XP_003490073) deposited in GenBank by using BioEdit 7.0.9. The BlTre-2 cDNA sequence was deposited in the NCBI GenBank (accession number MZ292465). The signal peptides, molecular mass and theoretical isoelectric point (pI), N-glycosylation sites and transmembrane helices of the deduced amino acid sequences of BlTre-2 were predicted by using the SignalP 4.1 Server, the ExPASy Compute pI/Mw tool (https://web. expasy.org/compute_pi/), the NetNGlyc 4.0 Server, and the TMHMM Server v. 2.0, respectively. The deduced amino acid sequences of BlTre-2 were compared with trehalase genes of other species available in GenBank. Multiple alignments of trehalase genes were conducted using the software Clustal X2. A phylogenetic tree was constructed based on the amino acid sequences by using the neighbor-joining (NJ) algorithm in MEGA 6.06. The reliability of the branching was tested using the bootstrap method (1,000 replications). Primer uses Primer name Primer sequence (5’-3’) The part fragment Tre-2-F GGACTGGAAAAGAGCGGTCA Tre-2-R GGTTTCAGATCGTCCTGGCACGATT 3’-RACE 3’-OGSP GTTCGACGATTTGGAACGTC 3’-OP TACCGTCGTTCCACTAGTGATTT 3’-IGSP ATACGCGGAACGTAATTGGC 3’-IP CGCGGATCCTCCACTAGTGATTTCACTATAGG 5’-RACE 5’-OGSP GGTCGAACGTGTTGTTGATG 5’-OP CATGGCTACATGCTGACAGCCTA 5’-IGSP GATCTGGTTCCTGGTCGGTG 5’-IP CGCGGATCCACAGCCTACTGATGATCAGTCGATG qRT-PCR Tre-1-F1 Tre-1-R1 AGTGAGTTTGCCTTCTGG GAGGTCTCGTGCTGTTGA Tre-2-F1 TCCTCGTTCGTTTGTGACGA Tre-2-R1 TTTGGCCAATTACGTTCCGC Reference gene Actin-88-F GCGCGACATTAAGGAGAAAC Actin-88-R CCATACCCAGGAAGGAAGGT Table 1. Primers used in this study. Jiamin Qin et al. – Molecular Characterization and Gene Expression of Trehalase Bombus lantschouensis4 BlTre-2 Expression in Different Tissues and Different Chronological Ages To clarify the distribution of BlTre-2 in B. lantschouensis, we dissected the workers in the ice-cold lysis buffer on ice. Ten tissues (antennae, head, muscle, leg, wing, integument, midgut, Malpighian tubule, fat body, and ovary) were collected from workers aged 15 days and placed separately into PCR tubes for RNA extraction. To get enough samples for RNA extraction, 3 to 15 workers were dissected (Table 2). Larvae, pupae, and adult workers aged 0 (new workers), 5, 10, 15, 20, 25, and 30 days old were dissected to investigate the relative expression of BlTre-2 at different chronological ages. A sample of workers of the same age from each of three different colonies was dissected to extract RNA from the whole body, and this triplicated. To determine the absolute copy of the target transcript, the cDNA template was diluted (10-1, 10-2, 10-3, 10-4, 10-5, 10-6, and 10-7) to gradient concentrations and then used to generate a standard curve. The qRT-PCR amplification reaction system contained 2 μL cDNA template, 0.8μL of each primer (10 μmol/L), 10 μL SYBR® Premix Ex TaqTM II, and 0.4 μL ROX reference dye and was finally topped to 20 μL volume with ddH2O. The amplification conditions were as follows: 94°C for 30 s followed by 40 cycles of 94°C for 5 s and 60°C for 30 s. Each sample was replicated three times. Actin-88 gene (Table 1) served as an endogenous reference gene for the determination of targeted mRNA for its continuously expressed in bumblebee (Li et al., 2010). completed, RNA was extracted from three workers exposed to the same treatment. To investigate the relative expression of the BlTre-1 and BlTre-2 genes, we designed two pairs of specific primers: Tre-1-F1 and Tre-1-R1; Tre-2-F1 and Tre- 2-R1 (Table 1) based on the conserved amino acid sequences of the two known forms of trehalase gene in B. lantschouensis (BlTre-1: GenBank accession nos. KJ025078; BlTre-2: GenBank accession nos. MZ292465), and the primers of the reference gene Actin-88-F and Actin-88-R (Li et al., 2010; Qin et al., 2015). The qRT-PCR reactions were conducted using an Mx3000 qPCR system (Agilent, USA) with buffers at 94°C for 30 s in 1 cycle; 94°C for 5 s and 60°C for 20 s in 40 cycles with a melt curve over a temperature ranging from 55 to 90°C. In each reaction, 25 μL of final volume was produced containing 10 μL of the SYBR® Premix Ex TaqTM II, 2 μL of cDNA sample, 0.8 μL of primer (10 μmol/L), 0.4 μL of ROX reference dye, and 11 μL of RNase-free and DNAase-free H2O, according to the manufacturer’s instructions of SYBR® Premix Ex TaqTM II Kit (Takara, Dalian, China). All samples were run in triplicate. The qRT-PCR values of the focal genes were normalized using the Actin-88 gene. Statistical Analyses Transcript quantifications were calculated using the 2-ΔΔCt (Livak & Schmittgen, 2001). The lowest expression levels in different tissues and at different chronological ages were stated as 1. All data were analyzed using LSD tests of one-way ANOVA in SPSS 13.0. Results Cloning and Characterization Analyses of BlTre-2 The BlTre-2 is 4,051 bp long, including an open reading frame (1,989 bp), a 5’-untranslated region (UTR) (1,307 bp), and a 3’-UTR (1,684 bp). The BlTre-2 transcript encoded a protein of 662 amino acids (about 76.81 kDa and an estimated pI of 5.94). The amino acids contained two signature motifs, PGGRFREFYYWDSY (residues 180-193) and QWDYPNAWPP (481-490), a highly conserved glycine- rich region, GGGGEY (545-550), a signal peptide of 30 amino acids and a cleavage site (CYA-ST) between 30 and 31 (Fig 1), and five putative N-glycosylation sites (amino acids 79, 276, 352, 386, and 527) suspected to be a glycoprotein. Residues 13-31 and 598-620 comprised two putative transmembrane domains, MLLSAAFLALLVVAPCYAS QVMTGILALVISLAAGFIGMVVY (Fig 1). The deduced amino acid sequence of trehalase from B. lantschouensis was aligned with the corresponding sequences of another insect trehalases (Fig 2). BlTre-2 is most similar to the other Hymenopterans, such as BtTre-2 (B. terrestris), BiTre-2 (B. impatiens), AmTre-2 (A. mellifera) and AfTre-2 (A. florea) (Table 3). It is also similar to Tre-1 and Tre-2 from Harpegnathos saltator, S. exigua, Bombyx mori, O. fuscidentalis, N. lugens, B. tabaci, and A. lucorum (Table 3). Tissue name Number of individuals Chronological Age Number of individuals Antennae 15 Larva 3 Head 5 Pupa 3 Muscle 3 Day 0 worker 3 Leg 5 Day 5 worker 3 Wing 15 Day 10 worker 3 Integument 5 Day 15 worker 3 Midgut 15 Day 20 worker 3 Malpighian tubule 15 Day 25 worker 3 Fat body Ovary 15 15 Day 30 worker 3 Table 2. The information of the number of individuals in each biological repetition for different tissues and chronological ages of B. lantschouensis. BlTre-1 and BlTre-2 Expression at 45°C and During Starvation In this study, 15-days old workers were exposed to 0, 1, 2, 3, 4, and 5 h at 45°C, representing the temperature treatments for our experiments. For the starvation treatments, the newly emerged (0 day) adult workers were starved for 0, 4, 8, 12, 16, 20, and 24 h. When the treatments were Sociobiology 68(4): e5443 (December, 2021) 5 The alignment of multiple sequences indicated that the insect Tre-2 gene is highly conserved, particularly in the middle of the putative catalytic domain (Fig 2). In addition, we used the amino acid sequences of selected trehalase genes to construct a phylogenetic tree, which shows that BlTre-2 has a higher identity with other Tre-2 genes in insects. The entire Tre-2 gene clustered together as a subgroup, and the Tre-1 gene clustered into another subgroup (Fig 3). Table 3. The similarity comparison between amino acid sequences of trehalase genes from B. lantschouensis and other insects. Species names Gene names GenBank No. Similarity to BlTre-1 Similarity to BlTre-2 Bombus lantschouensis BlTre-1 BlTre-2 KJ025078 MZ292465 – 55.7 % 55.7 % – Bombus terrestris BtTre-1 BtTre-2 XP_003400853 XP_003393687 99.5 % 56.0 % 55.2 % 98.9 % Bombus impatiens BiTre-1 BiTre-2 XP_003491166 XP_003490073 99.0 % 55.1 % 54.9 % 97.5 % Apis mellifera AmTre-1 AmTre-2 XP_393963 BAF81545 88.3 % 59.6 % 54.5 % 84.2 % Apis florea AfTre-1 AfTre-2 XP_003695047 XP_003696950 87.4 % 56.1 % 53.9 % 91.8 % Harpegnathos saltator HsTre-1 HsTre-2 EFN81352 EFN85130 72.1 % 56.2 % 53.5 % 85.8 % Spodoptera exigua SeTre-1 SeTre-2 ABY8628 ABU95354 58.5 % 56.2 % 54.1 % 69.4 % Bombyx mori BmTre-1 BmTre-2 NP_001037458 NP_001036910 60.4 % 57.3 % 51.7 % 68.6 % Omphisa fuscidentalis OfTre-1 OfTre-2 ABO20846 ABO20845 59.9 % 56.0 % 53.8 % 67.5 % Nilaparvata lugens NlTre-1 NlTre-2 ACN85420 ACN85421 60.9 % 54.2 % 54.4 % 72.9 % Bemisia tabaci BtTre-1 BtTre-2 AFV79626 AFV79627 57.1 % 57.7 % 50.4 % 70.4 % Apolygus lucorum AlTre-1 AlTre-2 AGK89789 AGL34007 58.3 % 58.0 % 65.0 % 71.2 % BlTre-2 Expression in Different Tissues and at Different Chronological Ages The qRT-PCR results detected the expression of BlTre-2 in 10 tissues and at various chronological ages of B. lantschouensis. The BlTre-2 had the highest expression levels in the ovary, followed by the midgut, antennae, muscle, Malpighian tubule, integument, wings, head, and fat bodies, with the lowest expression level in legs (Fig 4A). The BlTre-2 gene expression in the larval stage is higher than that in the pupal stage (P = 0.011). In addition, analysis of different chronological ages revealed that BlTre-2 had the lowest expression in 0-day-old workers, and highest expression in 30-day-old workers. From day 0 to 15, the gene expression increased gradually (Fig 4B). BlTre-1 and BlTre-2 Expression at 45°C and During Starvation The expression of BlTre-1 increased as temperature treatment time increased, reaching the highest level at 3 h, and then declined progressively (Fig 5A). BlTre-2 expression declined with treatment time in the first 3 h, and then it increased at the 4 h time point (Fig 5B). Overall, the results showed that in the 45°C treatments, the change of expression patterns differ between BlTre-1 and BlTre-2. In the starvation experiment, both BlTre-1 and BlTre-2 had the highest expression levels in adult bees starved for 16 and 20 h, as compared to other time points (Fig 5C, D). The expression levels of BlTre-1 in bees starved for 4 to 12 h and 24 h did not differ significantly from that of bees starved for 0 h (Fig 5C). However, the expression level of BlTre-2 in starved adult B. lantschouensis was higher than that in individuals starved for 0 h, and its expression increased with starvation time. Jiamin Qin et al. – Molecular Characterization and Gene Expression of Trehalase Bombus lantschouensis6 1 CATCGTTATCTTGCGAATTCGAAGCAAAAGAGGAACTTGTTAAAAGAAGGGAAGTAA 58 AGGAGACAAGAGAGAAGAAGAGGGATTGTGTGAAAGGGTCGACCGGTGGAGAAAAAA 115 atggcttggagctgcacgcgctgcggttcgacgaatatgctgctgagtgctgcgttcctc 1 M A W S C T R C G S T N M L L S A A F L 175 gcgcttctcgtcgttgctccgtgttacgctagcacagagaaggcaagctacgtgaaaccg 21 A L L V V A P C Y A S T E K A S Y V K P 235 cctccgtgtcagagcgatatttactgccatggcgagctgctgcacacgatacagatggcc 41 P P C Q S D I Y C H G E L L H T I Q M A 295 tcgatctacaaggactcgaagacgttcgtcgacatgaagatgaaattctcgccgaacgag 61 S I Y K D S K T F V D M K M K F S P N E 355 acgctgctcctatttcgcgaattcatggaaagcgtgaatcaaacaccgaccaggaaccag 81 T L L L F R E F M E S V N Q T P T R N Q 415 atcgaacaattcatcaacaacacgttcgaccaagaaggatccgagttcgaggaatggaac 101 I E Q F I N N T F D Q E G S E F E E W N 475 ccagtggactggaccagccaaccgaagtttcttaacaaaatccacgatcacgatcttcgc 121 P V D W T S Q P K F L N K I H D H D L R 535 aaatttgcctctgatttgaaccaaatttggaaaatgttgggacgaaagatgaaagacgac 141 K F A S D L N Q I W K M L G R K M K D D 595 gtgcgggtcaacgaggatcgatattccatcatctacgtgccgaatccggtgatcgtgccc 161 V R V N E D R Y S I I Y V P N P V I V P 655 ggcggccgattccgcgagttctactactgggactcgtactggatcgtgaaagggctgctg 181 G G R F R E F Y Y W D S Y W I V K G L L 715 ctttcggagatgtacaccaccgtcaaaggaatgttaaccaatttcgtctctctggtggac 201 L S E M Y T T V K G M L T N F V S L V D 775 aagatcggtttcatcccgaacggaggcagaatctactacgctaggagatctcagcctccc 221 K I G F I P N G G R I Y Y A R R S Q P P 835 atgttgattcctatggtcgaagagtatctgaaggtgaccatcgactacaaatgcctggag 241 M L I P M V E E Y L K V T I D Y K C L E 895 gataaccttcaccttctagagaaggagtttgaattttggatgaccaataggacggtggac 261 D N L H L L E K E F E F W M T N R T V D 955 gttgaagtggatggagtgaagtacactttagccagattcttcgaggagtcttcgggacct 281 V E V D G V K Y T L A R F F E E S S G P 1015 cgaccagaatcctacaaagaggattacctgaccagccaaagttttcgcacgaacgaagag 301 R P E S Y K E D Y L T S Q S F R T N E E 1075 aaggacaactattacgcggaattgaagaccgcggccgagtccggctgggacttttctagt 321 K D N Y Y A E L K T A A E S G W D F S S 1135 cgatggttcatactagacggcacgaacaaaggtaacctgacgaacttgaaaacgagatac 341 R W F I L D G T N K G N L T N L K T R Y 1195 attgtccccgtggacttgaattcgataatatatcgaaacgcgcagctgctagaacagtac 361 I V P V D L N S I I Y R N A Q L L E Q Y 1255 aatcaaaggatgggcaacgagaccaaggccgcgtattaccggaaaagagcggaggactgg 381 N Q R M G N E T K A A Y Y R K R A E D W 1315 aaaagagcggtcacggccgtactgtggcacgatgaagtcggtgcttggctcgactacgat 401 K R A V T A V L W H D E V G A W L D Y D 1375 ttactgaacgacatcaaaagagattatttttatccgacgaacgttctgccgctttggacc 421 L L N D I K R D Y F Y P T N V L P L W T 1435 gattgttacgacatcgcaaagagagaggaatacatagcgaaggtgctcaagtatctagag 441 D C Y D I A K R E E Y I A K V L K Y L E 1495 aaaaatcaaataatgttaaatttgggcggtataccgaccaccctcgaacactctggtgaa 461 K N Q I M L N L G G I P T T L E H S G E Fig 1. Nucleotide and deduced amino acid sequences of BlTre-2. The numbers on the left are the positions of nucleotides and amino acids in the sequences; underlined amino acid residues represent the signal peptide; the cleavage site is indicated by an arrow; the N-glycosylation sites are indicated by a box; the highly conserved glycine-rich region is shaded gray and the trehalase signature motif is shaded red; transmembrane domains are gray and boxed. Sociobiology 68(4): e5443 (December, 2021) 7 1555 caatgggattacccgaatgcctggccgcccttgcaatactttgtcatcatgtcgttgaat 481 Q W D Y P N A W P P L Q Y F V I M S L N 1615 aacaccggagacccgtgggcgcagaggctcgcctacgagatcagccaacgatgggttcgc 501 N T G D P W A Q R L A Y E I S Q R W V R 1675 agcaactggaaggcgttcaacgagacgcacagcatgttcgagaagtatgacgccacggta 521 S N W K A F N E T H S M F E K Y D A T V 1735 tcaggcggtcacggaggtggcggtgagtacgaggtgcaactaggtttcggttggagcaac 541 S G G H G G G G E Y E V Q L G F G W S N 1795 gggatcatcatggacttgctgaacaagtacggagatagactgacagccgaaattttcctc 561 G I I M D L L N K Y G D R L T A E I F L 1855 gccatagtgcagagcttggcccctccagccgtcgtcgtttcgaccgccggtcaagtgatg 581 A I V Q S L A P P A V V V S T A G Q V M 1915 accggtattctcgccctcgtaatatcgttggccgcgggattcatcggaatggtggtttac 601 T G I L A L V I S L A A G F I G M V V Y 1975 aaaaggcgacactactatgttcctggaccatcgacgatgccaaacaagagaaaagtgatc 621 K R R H Y Y V P G P S T M P N K R K V I 2035 tcaccgaccggaaacgtttatcgaaagaggatcgcctacactgaattgaaggacatgaac 641 S P T G N V Y R K R I A Y T E L K D M N 2095 aatgattgaCGACCGTTGCTCTTTTAGAAAGCCGATTCGTTTAAAGAGACTCTTAAAGCG 661 N D * 2155 CAACGAGAGCACAAGAAGACCGGGGATAGGATAAAAGGAGCGCAGACGCAAAAAGGACAC 2215 CAACTAGAAACCAGAAACCGTCGATTACTGACTGATCGCGATCAAGATCGACCGTGAACC 2275 AACCAACGAAGATCGTCGCTTTCGATTTTCTCGCCAAAGGCTAGAAAGCTTGGTAACAAT 2335 TCGTGTGCGGTTTCAGATCGTCCTGGCACGATTCGACTACGCGAACATCATCGCCGTTGA 2395 TTCGCGCACGCATCTATCGCGGTGAATCTTCCGACTCGAACGTTTTTACGGTCGGATATT 2455 CGTAACAAAAGTTCGCACGTTGGTCGTGCGTGATCACGTCCGTCTCAAGTTCTTTTTCTC 2515 TTTCTCGGTTTTCAATTCGGTAAGTGGATCGCGCGCGCTACATTCGCGGGGAAGGTGATA 2575 CGGTAGCACGGTGACACGTTAACGACTCTTTGTAGGACAACTCTGTCGCTGATAGACTAA 2635 AGCGCGAAAAATCGACTTCTCCGAAGGAAAACGTTCGAGAAGAGACGGCAGTGCGGAACG 2695 ATTTGGAACGCGACACAGCACGGCACTAAAAACCGCTGGAAACCGTGCGTCTCGCTCGCC 2755 AAGCTTCGCCGCGACTTCGACGGGTGTCCGCTCGCGTCGTGGGCTCCCCCACTTTAACCC 2815 TTCCCTTCCACTACGCGCTCGGCCTAATTACACGACAGTTATCTTCATTGCAAGCACGCG 2875 CTTATCCTTCTCGAATTACCTTATAATTTCGAAATACACTGGCGACGAGCGAAACTCGCA 2935 AAATTGAACACGAATGATGGTGTGAAGAGCTGTGCAAGGATCGGAACGAGCAACAACGTG 2995 CGTGCGTTGAAGCTCGGATGAAGAATGTGCAACTTTGCGACCAAGCTTTGTGGCACGTAC 3055 TTCCACGTTCATTGTTTATAGGTGAGTTTAGATAATCGATTCTGTAGATAAAAATGTCGT 3115 ATCTTTGAGGATATTTTTGTATAAAGAGCAGCTTGTACGTTTAGTGAAAGTCGTGTTCTT 3175 ATTGCTATTTACCATCCGGGATAATTCGCTTGAACGTTATTATCGACTTGTATAAACGAG 3235 TCGCGCAAAGTTGCCGCAAGGTGCGCATTGCTATCCGGAAACGGTGACGAGATGAACGAT 3295 CAATTACCGACGACGAGATTTCGCAAGAACCGGTCTTCGTGCTTTGTATCCGTCTAATTG 3355 TAAAAGATGCATCGAGCTATCCTCGTTCGTTTGTGACGACCGAATCGAAACAAGAGACGT 3415 CGTTTCTAATCCGAACTCTATGCTCGCTCAACTTTTCTTGTAAATAGTAATACGTGTTTT 3475 CGTTGTTCGACGATTTGGAACGTCGATACGCGGAACGTAATTGGCCAAACGCATAAGACA 3535 ATTTGAACTGTTACTTCTACTATAATCGTTGTTTATAGACAGATTTACGTATCAAAAAGC 3595 TCGAACGAACAAAATTTTGCACGGCCACGGAACGAGTTATTGACGAACCGTCGTCAAAGC 3655 GCGTCAACCGACGAATCGAAAATCGACTGGTTCGAGACTGAAGAACGTTACCGCGATATC 3715 TGCATCTTCCGCTAATCGCGATCGATCGCCGGCCGACGAGCATCGACCAACGAGCACCGC 3775 GTCTTAATGATTTTTGACACTGTTAGTTTTATTGTGAGCGACGCGAATATTATATATATA 3835 CAGATATATATATATAGATAGATAGATAGTTAGGTAGATAGATAGAGAATTTTGTGTTTG 3895 CGAGTGAACGAAAAATCGGTCGAGAGCCGACGAAGAATGATCGCGGTTGATCCTATGAGG 3955 AAATTTACCGCATCACGAAGGATGACGACGCGGTCGGATGGACTCGATCGTTAGCTGCGA 4015 CTAAAAAAAAAAAAAAAAAACCTATAGTGAAATCACT Fig 1. Nucleotide and deduced amino acid sequences of BlTre-2. (Continuation) Jiamin Qin et al. – Molecular Characterization and Gene Expression of Trehalase Bombus lantschouensis8 Fig 2. Comparison of the amino acid sequences of Tre in B. lantschouensis with those in other orthologs. Letters on the left and numbers on the right are the gene names and positions of amino acids on the sequences, respectively. The conserved and similar amino acid residues are labeled in black and gray backgrounds, respectively. The highly conserved glycine-rich regions of trehalase are indicated by a double underline.     BlTre-2 B. lantschouensis MAWSC-TRCGSTNMLLSAA--FLALLVVAPCYASTEKASYVKPPPCQSDIYCHGELLHTIQMA 60 BtTre-2 B. terrestris MAWSC-TRCGSTNMLLSAA--FLALLVVAPCYASTEKASYVKPPPCQSDIYCHGELLHTIQMA 60 BiTre-2 B. impatiens MACSC-TRCGSTNMLLSAV--FLAFLVVAPCYASTEKASYVKPPPCQSDIYCHGELLHTIQMA 60 AmTre-2 A. mellifera MASSCSIRCGSRNILVNAAATFLALLVVLRCFANAE-----KPSPCQSDVYCRGELLHTIQMA 58 AfTre-2 A. florea MASSCSIRCGSRNILVNAATTFLALLVVLRCFANAE-----KPPPCQSDVYCRGELLHTIQMA 58 BlTre-1 B. lantschouensis --------MSSGLLIAVGVIGLIAALTDAASIGHAS----VKATDCYSEIYCTGELLKTVQLS 51 BtTre-1 B. terrestris --------MPSGLLIAVGVIGLIAALTDAASIGHAS----VKATDCYSEIYCTGELLKTVQLS 51 BiTre-1 B. impatiens --------MPSGLLIAVGVIGLIAALTDAASIGHAS----VKATDCYSEIYCTGELLKTVQLS 51 AmTre-1 A. mellifera --------MMPGLFAFLGVA-LIASLTDAASIRRAN----RKAMDCYSEIYCTGELLKTIQLA 50 AfTre-1 A. florea -------MQAAGVFAFLGVA-LIASLTDAASIRRAS----RKAMDCYSEIYCTGELLKTIQLA 51 BlTre-2 B. lantschouensis SIYKDSKTFVDMKMKFSPNETLLLFREFMESVNQTPTRNQIEQFINNTFDQEGSEFEEWNPVD 123 BtTre-2 B. terrestris SIYKDSKTFVDMKMKFSPNETLLLFREFMESVNQTPTRNQIEQFINNTFDQEGSEFEEWNPVD 123 BiTre-2 B. impatiens SIYKDSKTFVDMKMKYSPNETLLLFREFMERVDQAPTRNQIEQFINNTFDQEGSEFEEWNPVD 123 AmTre-2 A. mellifera SIYKDSKTFVDMKMKRPPDETLKSFREFMERHEQMPTRYQIERFVNDTFDPEGSEFEDWDPDD 121 AfTre-2 A. florea SIYKDSKTFVDMKMKHPPHETLKLFREFMDRHDQMPTRHQIERFVNDTFDPEGSEFEEWDPDD 121 BlTre-1 B. lantschouensis NIYSDSKTFVDLQQINDPEITLANFYELMKETNNKPTKSQLIQYVNENFIS-SSELVNWTLSD 113 BtTre-1 B. terrestris NIYSDSKTFVDLQQINDPEITLANFYELMKETNNKPTKSQLIQYVNENFIS-SSELVNWTLSD 113 BiTre-1 B. impatiens NIYSDSKTFVDLQQINDPEITLANFYELMKETNNKPTKSQLTQYVNENFVA-SNELVNWTLSD 113 AmTre-1 A. mellifera EIFPDSKTFVDLHQMNDPEITLSNFYSLMNETGNKPSKSQLARYVNENFAS-SNELVNWTLPD 112 AfTre-1 A. florea EIFPDSKTFVDLHQINDPEITLSNFYSLMNETGNKPSKSQLTRYVNENFAS-SNELVNWTLSD 113 BlTre-2 B. lantschouensis WTSQPKFLNKIHDHDLRKFASDLNQIWKMLGRKMKDDVRVNEDRYSIIYVPNPVIVPGGRFRE 186 BtTre-2 B. terrestris WTSQPKFLNKIHDHDLRKFASDLNQIWKMLGRKMKDDVRVNEDRYSIIYVPNPVIVPGGRFRE 186 BiTre-2 B. impatiens WTSQPKFLNKIHDHDLRKFASDLNQIWKMLGRKMKDDVRINEDRYSIIYVPNPVIVPGGRFRE 186 AmTre-2 A. mellifera WTFRPKFLSRILDDDLRNFASELNGIWKMLGRKMKDDVRVNEELYSIIYVPHPVIVPGGRFRE 184 AfTre-2 A. florea WTFRPKFLSRILDDDLRNFASDLNSIWKMLGRKMKDDVRVNEELYSIIYVPNPVIVPGGRFRE 184 BlTre-1 B. lantschouensis WTNNPSILQRIQEPKYYEWAKDLNEIWKKLARKVNPEVARQPDRHSLIYVPNGLIIPGGRFKE 176 BtTre-1 B. terrestris WTNNPSILQRIQEPKYYEWAKDLNEIWKKLARKVNPEVARQPDRHSLIYVPNGLIIPGGRFKE 176 BiTre-1 B. impatiens WTNNPSILQRIQEPKYYEWVKDLNEIWKKLARKVNPEVARQPDRHSLIYVPNGLIIPGGRFKE 176 AmTre-1 A. mellifera WTESPSILKRINEAKYREWAKHLNEIWKELARKINPEVAEYPERHSLIYVDNGFIVPGGRFKE 175 AfTre-1 A. florea WTENPSILKRINEAKYREWAKHLNEIWKELARKINPEVAEYPERHSLIYVNNGFIVPGGRFKE 176 BlTre-2 B. lantschouensis FYYWDSYWIVKGLLLSEMYTTVKGMLTNFVSLVDKIGFIPNGGRIYYARRSQPPMLIPMVEEY 249 BtTre-2 B. terrestris FYYWDSYWIVKGLLLSEMYTTVKGMLTNFVSLVDKIGFIPNGGRIYYARRSQPPMLIPMVEEY 249 BiTre-2 B. impatiens FYYWDSYWIVKGLLLSEMYTTVKGMLTNFVSLVDKIGFIPNGGRIYYARRSQPPMLIPMVEEY 249 AmTre-2 A. mellifera FYYWDSYWIVKGLLLSEMYTTVKGMLTNFVSLVDKIGFIPNGGRIYYTMRSQPPMLIPMVDEY 247 AfTre-2 A. florea FYYWDSYWIVKGLLLSEMYTTVKGMLSNFVSLVDKIGLIPNGGRIYYVMRSQPPMLISMVDEY 247 BlTre-1 B. lantschouensis FYYWDSYWVIEGLLLSDMYQTARGMIDNFLYMVQKYGFIPNGGRIYYLMRSQPPLIHLMVSKY 239 Sociobiology 68(4): e5443 (December, 2021) 9 BtTre-1 B. terrestris FYYWDSYWVIEGLLLSDMYQTARGMIDNFLYMVQKYGFIPNGGRIYYLMRSQPPLIHLMVSKY 239 BiTre-1 B. impatiens FYYWDSYWVIEGLLLSDMYQTARGMIDNFLYMVQKYGFIPNGGRIYYLMRSQPPLIHLMVSKY 239 AmTre-1 A. mellifera FYYWDSYWVIEGLLLSDMYQTARGMIDNFLYMVKKYGFIPNGGRIYYLMRSQPPLLHLMVSRY 238 AfTre-1 A. florea FYYWDSYWVIEGLLLCDMYQTARGMIDNFLYMVKKYGFIPNGGRIYYLMRSQPPLLHLMVSRY 239 BlTre-2 B. lantschouensis LKVTIDYKCLEDNLHLLEKEFEFWMTNRTVDVEVDGVKYTLARFFEESSGPRPESYKEDYLTS 312 BtTre-2 B. terrestris LKVTNDYTWLEDNLHLLEKEFEFWMTNRTVDVEVDGVKYTLARFFEESSGPRPESYKEDYLTS 312 BiTre-2 B. impatiens LKVTNDYKWLEDNLHLLEKEFEFWMTNRTVDVEVDGVRYTLARFFEESSGPRPESYKEDYLTS 312 AmTre-2 A. mellifera LKITHDYEWLENNLYLLEKEFDFWMTNRTVEIEVDGVNYVLARYNEQSSGPRPESYKEDYLTS 310 AfTre-2 A. florea LKTTHDYEWLENNLYLLEKEFDFWMTNRTVEIEVDGVNYVMARYNEESSGPRPESYKEDYLTS 310 BlTre-1 B. lantschouensis LDFTGDYDYLRKVIPTLESEFAFWQQKRMIDVKKNGRTYKMGHYAVNSTRPRPESYREDYEQA 302 BtTre-1 B. terrestris LDFTGDYDYLRKVIPTLESEFAFWQQKRMIDVKKNGRTYKMGHYAVNSTRPRPESYREDYEQA 302 BiTre-1 B. impatiens LDFTGDYDYLRKVIPTLESEFAFWQQKRMIDVKKNGRTYKMGHYAVNSTRPRPESYREDYEQA 302 AmTre-1 A. mellifera LDFTGDYDYLRSIISTLETEFSFWQREKMIDVEKDGKIYKMAHYVVNSTSPRPESYREDYLMA 301 AfTre-1 A. florea LDFTGDYDYLRSIISTLETEFSFWQREKMIDVEKDGKIYKMAHYMVNSTSPRPESYREDYLMA 302 BlTre-2 B. lantschouensis QSFRTNEEKDNYYAELKTAAESGWDFSSRWFILDGTN-KGNLTNLKTRYIVPVDLNSIIYRNA 374 BtTre-2 B. terrestris QSFRTNEEKDNYYAELKTAAESGWDFSSRWFILDGTN-KGNLTNLKTRYIVPVDLNSIIYRNA 374 BiTre-2 B. impatiens QSFRTNEEKDNYYAELKTAAESGWDFSSRWFILDGTN-KGNLTNLKTRYIIPVDLNSIIYRNA 374 AmTre-2 A. mellifera QSFRTNEEKDNYYSELKTAAESGWDFSSRWFILDGTN-KGNLTNLKTRYIIPVDLNSIIYRNA 372 AfTre-2 A. florea QSFRTNEEKDNYYAELKTAAESGWDFSSRWFILDGTN-KGNLTNLKTRYIIPVDLNTIIYRNA 372 BlTre-1 B. lantschouensis QLLPE-KSRDFFYNNIKAGAESGWDFSNRWCIADNNNRTLSLLNISTQHIIPVDLNAILQQNA 364 BtTre-1 B. terrestris QLLPE-KSRDFFYNNIKAGAESGWDFSNRWCIADNNNRTLSLLNISTQHIIPVDLNAILQQNA 364 BiTre-1 B. impatiens QLLPE-KSRDFFYNNIKAGAESGWDFSNRWCIADNNNRTLSLLNISTQHIIPVDLNAILQQNA 364 AmTre-1 A. mellifera QRIPE-KSRDFFYNNIKAGAESGWDFSNRWFIRNNNSSTLSLYNISTQYIIPVDLNAILQQNA 363 AfTre-1 A. florea QRIPE-KSRDXFYNNIKAGAESGWDFSNRWFIRNNNSSALSLYNISTQYIIPVDLNAILQQNA 364 BlTre-2 B. lantschouensis QLLEQYNQRMGNETKAAYYRKRAEDWKRAVTAVLWHDEVGAWLDYDLLNDIKRDYFYPTNVLP 437 BtTre-2 B. terrestris QLLEQYNQRMGNETKAAYYRKRAEDWKRAVTAVLWHDEVGAWLDYDLLNDIKRDYFYPTNVLP 437 BiTre-2 B. impatiens QLLEQYNQRMGNETKAAYYRKRAEDWKRAVTAVLWHDEVGAWLDYDLLNDIKRDYFYPTNVLP 437 AmTre-2 A. mellifera VLLAQYNQRMGNESKVAYYQKRAAEWKRAIQAVLWHDEVGAWLDYDILNDIKRDYFYPTNILP 435 AfTre-2 A. florea MLLAKYNQRMGNESKVAYYQKRAAEWKRAITAVLWHEEVGVWLDYDMLNDIKRDYFYPTNILP 435 BlTre-1 B. lantschouensis RLLGEFHSLLGNNAKSQYYHKVASQLQMAIDNVLWNEEEGTWLDYDMKNEKPRHAFYPSNLAP 427 BtTre-1 B. terrestris RLLGEFHSLLGNNAKSQYYHKVASQLQMAIDNVLWNEEEGTWLDYDMKNEKPRHAFYPSNLAP 427 BiTre-1 B. impatiens RLLGEFHSLLGNNAKSQYYHKVASQLQMAIDNVLWNEEEGTWLDYDMKNAKPRHAFYPSNLAP 427 AmTre-1 A. mellifera RLLGEFHTLLGNNAKSQYYQKIASQLQTAIDNILWNEADGIWLDYDLKNQRPRHMFYPSNLAP 426 AfTre-1 A. florea RLLGEFHTLLGNNAKSQYYQKIASQLQTAIDNVLWNEADGIWLDYDMKNQRPRHMFYPSNLAP 427 BlTre-2 B. lantschouensis LWTDCYDIAKREEYIAKVLKYLEKNQIMLNLGGIPTTLEHSGEQWDYPNAWPPLQYFVIMSLN 500 BtTre-2 B. terrestris LWTDCYDIAKREEYIAKVLKYLEKNQIMLNLGGIPTTLEHSGEQWDYPNAWPPLQYFVIMSLN 500 BiTre-2 B. impatiens LWTDCYDIAKREEYIAKVLKYLEKNQIMLNLGGIPTTLEHSGEQWDYPNAWPPLQYFVIMSLN 500 AmTre-2 A. mellifera LWTDCYDIAKREEYVSKVLKYLEKNKIMLNLGGIPTTLEHSGEQWDYPNAWPPLQYFVIMALN 498 AfTre-2 A. florea LWTDCYDLAKREEYVSKVLKYLEKNKIMLNLGGIPSTLEHSGEQWDYPNAWPPLQYFVIMALN 498 Fig 2. Comparison of the amino acid sequences of Tre in B. lantschouensis with those in other orthologs. (Continuation) Jiamin Qin et al. – Molecular Characterization and Gene Expression of Trehalase Bombus lantschouensis10 BlTre-1 B. lantschouensis LYTRSYNRLQRKRYALSIVKYLKTQNIDTFLGGTPTSLNYTGEQWDFPNAWPPLQSFIVMGLY 490 BtTre-1 B. terrestris LYTRSYNRLQRERYALSIVKYLKTQNIDTFLGGTPTSLNYTGEQWDFPNAWPPLQSFIVMGLY 490 BiTre-1 B. impatiens LYTRSYNRLQRERYALSIVKYLKTQNIDTFLGGTPTSLNYTGEQWDFPNAWPPLQSFIVMGLY 490 AmTre-1 A. mellifera LYTKSYNRGQREYYGAATLRYLKSQNIDNFFGGTPTSLNHTGEQWDFPNAWPPLQSFIVMGLH 489 AfTre-1 A. florea LYTKSYNRGQREHYGATTLRYLKSQNIDSFFGGTPTSLNHTGEQWDFPNAWPPLQSFIVMGLH 490 BlTre-2 B. lantschouensis NTGDPWAQRLAYEISQRWVRSNWKAFNETHSMFEKYDATVSGGHGGGGEYEVQLGFGWSNGII 563 BtTre-2 B. terrestris NTGDPWAQRLAYEISQRWVRSNWKAFNETHSMFEKYDATVSGGHGGGGEYEVQLGFGWSNGII 563 BiTre-2 B. impatiens NTGDPWAQRLAYEISQRWVRSNWKAFNETHSMFEKYDATVSGGHGGGGEYEVQLGFGWSNGII 563 AmTre-2 A. mellifera KTEDPWAQRLAYEISERWVRSNYKAYNETHSMFEKYDATVSGGHGGGGEYEVQLGFGWSNGVI 561 AfTre-2 A. florea NTEDPWAQRLAYEISERWVRSNYKAYNETHSMFEKYDATVSGGHGGGGEYEVQLGFGWSNGVI 561 BlTre-1 B. lantschouensis WTGVEEAVNFAHELAFRWLGSNYAGYVEYKEMFEKYDSLTPGKSGGGGEYDVQSGFGWANGVV 553 BtTre-1 B. terrestris WTGVEEAVNFAHELAFRWLGSNYAGYVEYKEMFEKYDSLTPGKSGGGGEYDVQSGFGWTNGVV 553 BiTre-1 B. impatiens WTGVEEAVNFAHELAFRWLGSNYAGYVEYKEMFEKYDSLTPGKSGGGGEYDVQSGFGWTNGVV 553 AmTre-1 A. mellifera WTGVREAMDFAHELAFRWLAANYAGYKETGQMFEKYDSIVPGQGGGGGEYNVQTGFGWTNGVV 552 AfTre-1 A. florea WTEAREAMDFAQELAFRWLSANYAGYKETGQMFEKYDSIVPGQGGGGGEYNVQTGFGWTNGVV 553 GGGGEY BlTre-2 B. lantschouensis MDLLNKYGDRLTAEI-FLAIVQSLAPPAVVVS-TAGQVMTGILALVISLAAGFIGMVVYKRRH 624 BtTre-2 B. terrestris MDLLNKYGDRLTAED-RFVIVQSLAPPAVVVS-TAGQVMTGILALVISLAAGFIGMVVYKRRH 624 BiTre-2 B. impatiens MDLLNKYGDRLTAED-RFVIVQSLAPPAVVVVSTAGQVMTGILALVISLAAGFIGMVVYKRRH 625 AmTre-2 A. mellifera MDLLNRYGDKLTAEDRFVATFHSNSTPQPVVVSTAGQVMTGILALVISLAAGFIG-------- 616 AfTre-2 A. florea LDLLNRYGDKLTAEDRFVATFHSNSTPQPVVVSTAGQVMTGILALVISLAAGFIGMVVYKRRH 624 BlTre-1 B. lantschouensis LEFLNTFPNIKVKEISYINDINTENRQ------------------------------------ 580 BtTre-1 B. terrestris LEFLNTFPNIKVKEISYINDINTEIRQ------------------------------------ 580 BiTre-1 B. impatiens LEFLNTFPNIKVKEISYINDINTEIRQ------------------------------------ 580 AmTre-1 A. mellifera LEFLNTFSSIKVREVGYEDDL-TEVEQ------------------------------------ 578 AfTre-1 A. florea LEFLNTFSTIKVREVGYEDDL-TEVEQ------------------------------------ 579 BlTre-2 B. lantschouensis YVPGPSTMPNKRKVISPTGNVYRKRIAYTELKDMNND 662 BtTre-2 B. terrestris YVPGPSTMPNKRKVISPTGNVYRKRIAYTELKDMNND 662 BiTre-2 B. impatiens YVPGPSTMPNKRKVISPTGNVYRKRIAYTELKDMNND 663 AmTre-2 A. mellifera ----------------------KMRCANNAAQ----- 626 AfTre-2 A. florea YVPGPSTMPNKRKVISPSGNLYRKRIAYTELKDMNNY 662 BlTre-1 B. lantschouensis -------------------------------------- BtTre-1 B. terrestris -------------------------------------- BiTre-1 B. impatiens -------------------------------------- AmTre-1 A. mellifera -------------------------------------- AfTre-1 A. florea --------------------------------------   Fig 2. Comparison of the amino acid sequences of Tre in B. lantschouensis with those in other orthologs. (Continuation) Sociobiology 68(4): e5443 (December, 2021) 11 48 Fig 3. Phylogenetic analysis of trehalase amino acid sequences from various species. B. lantschouensis (BlTre-1: KJ025078); B. terrestris (BtTre-1: XP_003400853; BtTre-2: XP_003393687); B. impatiens (BiTre-1: XP_003491166; BiTre-2: XP_003490073); A. florea (AfTre-1: XP_003695047; AfTre-2: XP_003696950); A. mellifera (AmTre-1: XP_393963; AmTre-2: BAF81545); Acyrthosiphon pisum (ApTre-1: XP_001956264; ApTre-2: XP_001949459); Laodelphax striatella (LsTre-1: AFL03409; LsTre-2: AFL03410); A. lucorum (AlTre-1: AGK89789; AlTre-2: AGL34007); B. mori (BmTre-1: NP_001037458; BmTre-2: NP_001036910); S. frugiperda (SfTre-1: ABE27189; SfTre-2: ACF94698); L. migratoria (LmTre-1: ACP28173); T. molitor (TmTre-1: AGO32658); Megachile rotundata (MrTre-1: XP_003705482). Discussion In our study, we cloned the BlTre-2 gene from B. lantschouensis using the homologous cloning and RACE techniques. The deduced amino acid sequence shared similarities with the Tre-1 and Tre-2 genes from various species, including a signal peptide leader, a glycine-rich region (GGGGEY), two signature motifs (PGGRFREFYYWDSY and QWDYPNAWPP), and putative N-glycosylation sites (Santos et al., 2012). Functions of the most conservative residues and regions remain unknown. Additionally, only one transmembrane domain has been found in most insects, including B. mori (Mitsumasu et al., 2005), Nasonia vitripennis (Tang et al., 2012), N. lugens (Gu et al., 2009), O. fuscidentalis (Tatun et al., 2008), S. exigua (Chen et al., 2010), T. castaneum (Tang et al., 2012), L. migratoria (Liu et al., 2016), and C. medinalis (Tian et al., 2016). However, the BlTre-2 gene contained two putative transmembrane domains, MLLSAAFLALLVVAPCYAS and QVMTGILALVISLAAGFIGMVVY (Fig 1), which are like those of A. mellifera (Lee et al., 2007), Laodelphax striatellus (Zhang et al., 2010), Spodoptera frugiperda (Silva et al., 2009), Jiamin Qin et al. – Molecular Characterization and Gene Expression of Trehalase Bombus lantschouensis12 and Aedes aegypti (Tang et al., 2012). However, previous research suggested that BlTre-1 gene have no transmembrane domain (Qin et al., 2015). In this study, although the proteins encoded by BlTre-2 gene showed obvious similarity to B. terrestris (98.9%), B. impatiens (97.5%), and A. mellifera (84.2%), the protein encoded by BlTre-2 gene only showed 55.7% similarity to those encoded by BlTre-1 gene (Table 3). In insects, Tre-1 and Tre-2 are involved in many physiological processes (Mitsumasu et al., 2005; Shi et al., 2019). Su et al. (1994) indicated that Tre-2 can help transport sugars into oocytes of B. mori. Furthermore, it has been shown that trehalose, glycogen, and glucose can be stored in growing oocytes during the period of vitellogenesis of Rhodnius prolixus, (Santos et al., 2012). Similarly, in B. tabaci and R. prolixus, the expression levels of Tre-2 were much higher in the ovary than in other tissues (Santos et al., 2012; Wang et al., 2014). In our study, the BlTre-2 gene had the highest expression levels in the ovary and then in the midgut of B. lantschouensis (Fig 4A). These findings suggest that the BlTre-2 gene may play an important role in providing materials and energy for oocyte development in B. lantschouensis. The Tre-2 gene is distributed differently in different tissues in insects, and this may be linked to its function. Previous studies have shown that the Tre-2 gene has higher expression in the integument of Spodoptera litura (Zou et al., 2013), the wing bud of N. lugens (Zhang et al., 2017), and the gut of S. exigua (Tang et al., 2008) and Bactrocera dorsalis (Xie et al., 2013) as compared with other tissues. Fig 4. The relative expression levels of BlTre-2 in different tissues (A) and chronological ages (B) of B. lantschouensis. AN: Antennae; HE: Head; MU: Muscles; LE: Legs; WI: Wings; IN: Integument; MG: Midgut; MT: Malpighian tubules; FB: Fat body; OV: Ovary; LA: Larva; PU: Pupa; D0-D30: Day 0 to day 30 worker. Each value represents mean ± S.D., and different letters above bars indicate a significant difference (p-0.05). Fig 5. The relative expression levels of two trehalase genes under adverse conditions. The BlTre-1 and BlTre-2 relative expression in workers which were exposed to 45°C ambient temperature for 0, 1, 2, 3, 4 and 5 h (A, B). The BlTre-1 and BlTre-2 relative expression in workers which were starved for 0, 4, 8, 12, 16, 20 and 24 h (C, D). Each value represents the mean ± S.D., and different letters above the bars indicate significant differences (p-0.05). Sociobiology 68(4): e5443 (December, 2021) 13 In B. lantschouensis, the BlTre-2 gene may be involved in providing energy to the chitin synthesis process in the midgut or in supporting peristaltic movement of the midgut. These results suggest that the BlTre-2 gene may perform specific functions in different tissues. Our study found, for the first time, that BlTre-2 has the highest expression level in 30-day-old worker bees (Fig 4B), so we can assume that this expression level of BlTre-2 is associated with the biological behavior of B. lantschouensis. Older workers may depend mainly on BlTre-2 to break down extracellular trehalose (mainly from food). Next, the expression of BlTre-2 was significantly higher in the larvae than in the pupae. It is possible that BlTre-2 expression is involved in the feeding and development of larvae. In B. dorsalis, Tre-2 was found to be highly expressed in metabolic tissues at both the adult and larval stages (Xie et al., 2013). The results also revealed that expression of BlTre-2 in 0- to 20-day-old workers first increased and then declined with age (Fig 4B). This result may be due to the social division of labor in B. lantschouensis colonies. Both younger and older workers are engaged in various activities, such as helping the queen secrete wax, nesting, and nursing, and strong workers take part in foraging and guarding. Previous studies suggested that insect trehalase, including TRE-1 and TRE-2 play critical roles in energy supply, growth, metamorphosis, stress recovery, chitin synthesis, and flight by catalyzing the hydrolysis of trehalose to glucose in insects (Wyatt, 1967; Thompson, 2003; Shukla et al., 2014). Insects adapt to changes in environmental temperature and maintain their energy by regulating their own body temperature. A high temperature not only breaks the moisture balance of the internal environment and interferes with normal metabolism but it also causes body temperature to rise and affects enzyme activity and protein function in insects (Du et al., 2007). Previous studies showed that trehalase optimizes temperature in the range of 40–65°C (Zou et al., 2013; Shukla et al., 2014; Youngjin & Yonggyun, 2017). At a high temperature, the parasitic nematode Aphelenchoides besseyi improves its resistance by upregulating the trehalase gene (Chen et al., 2016). In our study, the expression levels of BlTre-1 and BlTre-2 were, respectively, higher and lower than at 0 h under the 45°C treatment conditions. We hold the opinion that the activity of soluble trehalase was high at 45°C because trehalose was gradually hydrolyzed into glucose and used for stress recovery. Bumblebees maintain vital activities by accumulating trehalose through soluble trehalase catalysis in such high-temperature conditions. By comparison, the expression level of BlTre-2 was lower than that of BlTre-1 for the 45°C treatment. This result suggests that BlTre-1 may be involved in providing energy for physiological activity and that BlTre-2 expression maybe constrained at a high temperature. Trehalose is a feedback-regulating substance involved in the feeding behavior and nutrient intake of insects (Wyatt, 1967). Trehalose provides energy when insects are starving; thus, it has a role in the regulation of insect functions under starvation conditions (Tang et al., 2014; Youngjin & Yonggyun, 2017). In H. axyridis adults, the stored food reserves can provide energy to sustain vital activities for 8 h, but energy limitations have a direct impact on the desire to find food (Tang et al., 2014). Our results showed no significant difference in the expression levels of both BlTre-1 and BlTre-2 in B. lantschouensis adults starved for 4 to 12 h as compared with those starved for 0 h, which may be because the stored food reserves were able to provide energy to sustain vital activities for 12 h in adults. In addition, the expression levels of BlTre-1 and BlTre-2 increased in adults starved for 16 to 20 h, which suggests that trehalose stores were degraded by trehalase. BlTre-1 and BlTre-2 function to facilitate the uptake and utilization of trehalose from blood and food, respectively (Yaginuma et al., 1996). Result of the present study show that BlTre-1 is a key gene involved in regulating energy metabolism and providing glucose at a high temperature. BlTre-1 and BlTre-2 might balance trehalose and provide energy during periods of starvation. Our study sheds light on the molecular function and gene expression of trehalase in B. lantschouensis, which adds further important information about the characteristics of this gene in the physiology and development of bumble bees in China. Different native populations in different regions of China may display different types of adaptations to coldness, which is associated with trehalase expression. We provide new knowledge to assist future selection and breeding of B. lantschouensis in China. Furthermore, The Tre-1 and Tre-2 genes participate in energy metabolism during developmental and physiological activities in various insects, which provides a reference for the protection and utilization of pollination insects. Acknowledgments This work was supported by the National Natural Science Foundation of China (31201858), the Central Public- interest Scientific Institution Basal Research Fund (Y2018PT66 and Y2021XK16), and Key Research and Development Project of Jiangxi Province (20192BBH80003). H.L.-B. is supported by the U.S. Department of Agriculture – National Institute of Food and Agriculture (USDA NIFA Award # NI181445XXXXG007). Authors Contribution JMQ: conceptualization, investigation, validation, methodology, writing. FL: investigation, methodology. JW: supervision, project administration. SYH: supervision. MI: review, resources. WL: resources. HML: writing. SDL: conceptualization, funding acquisition, supervision, project administration, writing. Jiamin Qin et al. – Molecular Characterization and Gene Expression of Trehalase Bombus lantschouensis14 References Ai, D., Cheng, S. H., Chang, H. T., Yang, T., Wang, G. R. & Yu, C. H. (2018). 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