Molecular Characterization and Expression of Vitellogenin and Vitellogenin Receptor of Thitarodes pui (Lepidoptera: Hepialidae), an Insect on the Tibetan Plateau

Molecular Characterization and Expression of Vitellogenin and Vitellogenin Receptor of Thitarodes... Vitellogenin (Vg) and vitellogenin receptor (VgR) play important roles in the vitellogenesis of insects. In this study, we cloned and characterized the two corresponding genes (TpVg and TpVgR) in an economically important insect, Thitarodes pui (Lepidoptera: Hepialidae), from the Tibetan plateau. The full length of TpVg is 5566 bp with a 5373 bp open reading frame (ORF) encoding 1,790 amino acids. Sequence alignment revealed that TpVg has three conserved domains: a Vitellogenin_N domain, a DUF1943 domain, and a von Willebrand factor type D domain (VWD). The full length of TpVgR is 5732 bp, with a 5397 bp ORF encoding 1798 amino acids. BLASTP showed that TpVgR belongs to the low-density lipoprotein receptor (LDLR) gene superfamily. Structural analysis revealed that TpVgR has a group of four structural domains: a ligand-binding domain (LBD), an epidermal growth factor (EGF)–precursor homology domain, a transmembrane (TM) domain, and a cytoplasmic domain. In addition, TpVgR has four cysteine-rich LDL repeats in the first ligand-binding site and seven in the second. Quantitative real-time polymerase chain reaction analysis revealed that the expression levels of TpVg and TpVgR are much higher in later pupa than in either the larval or adult stage, implying that the synthesis and uptake of Vg in T. pui occurs in the later pupal stage. These results will help us to understand the molecular mechanism of the reproductive capacity and will provide new insight into the mass rearing and utilization of T. pui. Key words: vitellogenin, vitellogenin receptor, Thitarodes pui Like other animals, insects require large amounts of vitellogenin (Vg) 1995, Cho and Raikhel 2001), Lepidoptera (Shu et  al. 2011, Qian to meet the nutritional needs of egg development. Vg, the precursor of et  al. 2015, Zhang et  al. 2016), Hemiptera (Upadhyay et  al. 2016), vitellin and the major yolk protein, is usually synthesized in the fat body, Coleoptera (Roy-Zokan et al. 2015), Hymenoptera (Chen et al. 2004), released into the hemolymph, and subsequently taken up by the vitel- and Blattaria (Tufail and Takeda 2007, 2008). The deduced amino logenin receptor (VgR) located on the external surface of competent acid (aa) sequences of these VgRs indicate that they are members of oocytes (Tufail and Takeda 2008, 2009). Vg has been studied exten- the low-density lipoprotein receptor (LDLR) family. In general, the sively in many insects of different orders. The identification of insect Vgs LDLR family members have five distinct, conserved motifs, including revealed that these Vgs belong to a large family of lipid transfer genes a ligand-binding domain (LBD) containing LDLR Class A  cysteine- and that they share similar structural motifs, including an N-terminal rich repeats, an epidermal growth factor (EGF)-like domain containing lipid binding domain (LPD_N), an unknown functional region LDLR Class B cysteine-rich repeats and YWXD repeats, an O-linked (DUF1943), and a von Willebrand factor type D similar domain (VWD) carbohydrate domain with a serine/threonine-rich track, a transmem- (Trewitt et al. 1992, Chen et al. 1994, Yano et al. 1994a, Veerana et al. brane (TM) domain, and a cytoplasmic tail (Tufail and Takeda 2007, 2014, Upadhyay et al. 2016, Zhao et al. 2016, Ibanez et al. 2017). 2009; Shu et al. 2011; Upadhyay et al. 2016; Zhang et al. 2016). VgR, the key element in the process of Vg uptake, plays an impor- Thitarodes pui (Lepidoptera: Hepialidae) is naturally dis- tant role during vitellogenesis. To date, the molecular characteristics tributed in Segrila Mountain of the Tibetan Plateau, which is of VgRs have been well documented in vertebrates and invertebrates characterized by a harsh climate with low temperatures and including many orders of insects, such as Diptera (Schonbaum et al. hypobaric hypoxia. T.  pui larvae are the hosts for the famous © The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/23/4924664 by Ed 'DeepDyve' Gillespie user on 16 March 2018 2 Journal of Insect Science, 2018, Vol. 18, No. 2 traditional Chinese medicinal caterpillar fungus, the ascomycete the pGEM-T vector (Takara, Dalian, China), and transformed into Ophiocordyceps sinensis (Berk.) Sung, Sung, Hywel-Jones, and Escherichia coli DH5α cells for amplification. Positive clones were Spatafora (syn. Cordyceps sinensis). assessed by PCR and sequenced. To obtain the full-length cDNA Once infection occurs, the fungus grows within the body cav- sequences of TpVg and TpVgR, a SMARTer RACE (rapid ampli- ity of the insect larvae and finally kills and mummifies them in fication of cDNA ends) cDNA Amplification Kit (Clontech) was their underground burrows. A  fruiting fungal body emerges from used according to the manufacturer’s instructions. The first round the front end of the caterpillar and protrudes from the ground in of RACE-PCR was performed using outer primer (Supplementary spring and early summer (Buenz et al. 2005). The medicinal activity Fig.  1 and Supplementary Table  1) and a universal primer mix is derived from the parasite complex of the mummified caterpillar (UPM) with the following protocol: one cycle of predenaturing at and the fungal stromata (Zhang et al. 2009). 94°C for 5 min,; 35 cycles of 30 s at 95°C, 30 s at 65°C, and 2 min The complete life cycle of T.  pui lasts at least 3 yr under natu- at 72°C; and a final extension at 72°C for 5 min. The second round ral conditions (Li et al. 2011). A better understanding of the molec- of RACE-PCR was performed using inner primers (Supplementary ular mechanisms regulating reproduction can indicate potential Fig.  1 and Supplementary Table  1) and a nested universal primer approaches for the mass rearing of these economically important (NUP). The second RACE-PCR protocol was performed as follows: insects. Vg and VgR are important molecular markers for insect one cycle of predenaturing at 94°C for 5 min; 35 cycles of 30 s at fecundity. In this study, we cloned the full lengths of vitellogenin 95°C, 30 s at 68°C, and 2 min at 72°C; and a final extension at 72°C (TpVg) and the vitellogenin receptor (TpVgR) of T.  pui, character- for 5 min. The RACE products were gel purified and sequenced as ized the molecular structure of the corresponding deduced proteins, described above. and analyzed their gene expression profiles at different developmen- tal stages. Our results are expected to provide the theoretical basis Sequence Comparisons and Phylogenetic Analysis for the conservation and utilization of this economically important The sequence similarities were analyzed using the online BLASP insect. program on the NCBI website (https://blast.ncbi.nlm.nih.gov/). The open reading frames (ORFs) of TpVg and TpVgR were pre- dicted using the NCBI ORF finder (https://www.ncbi.nlm.nih.gov/ Materials and Methods orffinder/). The molecular weights and isoelectric points (pIs) of Insect Rearing and Collection the deduced protein sequences were predicted using the ExPASy T.  pui samples were collected in the Tibetan Plateau Peculiar Bio- proteomics server (http://www.expasy.org). The domain architec- resources Research Station of Sun Yat-sen University at Nyingchi in ture and the conserved domains were analyzed using the online the Tibet Autonomous Region (4,156 m altitude, 29°36′N, 94°36′E) servers of Scan-Prosite (http://prosite.expasy.org/scanprosite/), from June to August in 2014. The larvae were reared as previously SMART (http://smart.embl-heidelberg.de/), and InterProScan described (Sun et al. 2011, Wu et al. 2015). The larvae of different (http://ebi.ac.uk/Tools/pfa/iprscan/). The signal peptide was pre- instars (third through eighth), female pupae of different ages (1, 9, dicted by using the SignalP 4.1 Server (http://www.cbs.dtu.dk/ 17, and 33 d old), and 1-d-old adult females were used for the ana- services/SignalP). The potential O-linked glycosylation sites were lysis of gene expression. All of the samples at different developmen- evaluated using the GPP Prediction Server (http://comp.chem.not- tal stages were freshly frozen in a Sample Protector (Takara, Tokyo, tingham.ac.uk/glyco/), and the TM regions were predicted using Japan) at −80°C until RNA isolation. the TMHMM server v2.0 (http://cbs.dtu.dk/services/TMHMM/). All the computational programs were run with the recommended default values set up by the programs. The phylogenetic tree was RNA Isolation and cDNA Synthesis constructed with MEGA 6.0 by using the neighbor-joining method Total RNA was extracted from the whole body of T.  pui using with a bootstrap of 1,000 replicates. TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions, and RNA purity and degradation were checked on 1% agarose gels and Bioanalyzer 2100 system (Agilent Technologies). Developmental Expression Profile of TpVg and For tissue-specific expression profiles, total RNAs were extracted TpVgR from the thorax and abdomen parts of newly emerged female adults Equal quantities of cDNA (1  µg) from different developmental which were stored separately in a refrigerator at −80°C. The RNA stages were used for a relative expression analysis of TpVg and sample was dissolved in 20 µl diethylpyrocarbonate (DEPC)-treated TpVgR messenger RNA (mRNA) using a real-time PCR machine H O and assessed by spectrophotometry (Nanodrop 2000, Thermo (LightCycler 480 real-time PCR machine, Roche Applied Science, Fisher Scientific, DE). A PrimeScript Reagent Kit with gDNA Eraser Switzerland) with SYBR Green (Invitrogen). β-actin was used as an (TaKaRa, Tokyo, Japan) was used to synthesize first-strand comple- internal control based on a previous study (Sun et al. 2011). Primers mentary DNA (cDNA) with 1µg total RNA in a 20 µl reaction mix- used for quantitative PCR (qPCR) are listed in Supplementary ture following the manufacturer’s recommendations. Table 1. The qPCR amplifications were conducted in a total volume of 10 µl, including 1 µg of cDNA, 5 µl of SYBR Green I Master mix, Molecular Cloning of TpVg and TpVgR 0.5  µl of each primer (10  µM), and a certain amount of ddH O. The qPCR program included one cycle of 95°C for 10 s, followed Two partial sequences of both Vg and VgR were identified from by 40 cycles of 95°C for 5 s, 60°C for 20 s, and 72°C for 20 s. To the pupal transcriptome of T. pui (unpublished data). The fragment ensure reliability, each sample included three biological replicates. sequences of TpVg and TpVgR were further verified by polymer - The melt curve genotyping analysis was performed to confirm the ase chain reaction (PCR) amplification using the primers listed in homogeneity of the PCR products. Reactions were performed in Supplementary Table  1. The PCR program was performed as fol- triplicate for each sample, and gene expression levels were normal- lows: one cycle of predenaturing at 95°C for 5 min; 32 cycles of 95°C −∆∆ ized against T.  pui β-actin according to the 2 CT method (Livak for 30 s, 53°C for 30 s, and 72°C for 2 min; and then 72°C for 5 min and Schmittgen 2001). for elongation. The PCR products were then gel purified, cloned into Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/23/4924664 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 2 3 cluster. Other VgRs from the same insect orders were also clustered. Results In the phylogenetic tree of VgRs, Lepidoptera VgRs formed a single Sequence and Structural Analysis of TpVg and branch and was separated from other species (Fig. 3). However, the TpVgR dendrogram of Vgs showed that the Vgs of Diptera and Lepidoptera The full sequence of TpVg was 5566  bp, including an 83  bp 5′ species were close together (Fig. 2), suggesting that the Vgs of those untranslated region (UTR), a 5373 bp ORF encoding 1,790 aa res- two insect orders share a closer ancestry than other insects. idues, and a 110  bp 3′ UTR (GenBank accession no. MF622538). The theoretical molecular weight was 204.43 kDa, and the pI was Expression Profile of TpVg and TpVgR 6.51. A BLASTP analysis showed that the aa sequence of TpVg had a The expression analysis of TpVg and TpVgR was conducted during 30 to 40% similarity with other insects from different orders. TpVg the larval, pupal, and adult stages by real-time PCR (Fig.  4). The showed a 34 % and 33 % similarity with Operophtera brumata and expression level of TpVg was normalized with a reference gene, Actias selene, respectively. As with other insects, TpVg in T. pui also β-actin of T.  pui. The results showed that TpVg had no detectable contained the typical conserved domains of the Vg protein. expression in the larval stage, but it had a high expression during Domain architecture analysis by SMART showed that the TpVg the pupae stage, decreasing significantly in adult females (Fig.  4). aa sequence has three conserved domains (Fig. 1): the vitellogenin_N The expression of TpVg began to increase in the pupal stage and domain, the DUF1943 domain, and the VWD. A signal peptide with reached its peak in the 25 and 33-d-old pupae, i.e., the final stage 18 amino acids (MRTFVLFALLATAFCANS) was found in the ana- of pupae. Then, the expression level of TpVg decreased significantly lysis of the predicted aa sequence of TpVg. In addition, the deduced in the adult stage. TpVgR had an expression profile similar to that aa sequences of TpVg included four N-glycosylation sites (NXS/T) of TpVg. TpVgR was found to have lower expression in the early and seven K/RXXR cleavage sites. pupal stage and then a remarkably high expression in the 17-d-old The full sequence of TpVgR was 5732  bp, including a 74  bp pupae, subsequently reaching a maximum expression level in the 5′ UTR, a 5397 bp ORF encoding 1,798 aa residues, and a 261 bp later pupae stage. Then, a lower expression level was maintained 3′ UTR (GenBank accession no. MF622539). The theoretical molec- in the adult period. In addition, the tissue-specific expression profile ular weight was 200.47 kDa, and the pI was 5.46. The analysis of the revealed that TpVg and TpVgR were highly expressed in the abdo- predicted aa sequence revealed that a signal peptide with 27 amino men which contained the reproductive organ-ovary (Fig. 5). acids (MGHADLLHIALIIVVNVFLLKWKMAGA) was located at the N-terminal of TpVgR. The protein sequence analysis indicated that TpVgR contained Discussion the characteristic features of the LDLR family (Fig.  1). TpVgR T. pui is a holometabolous insect with a long life history. The com- showed two LBDs with four class A  (LDLa) cysteine-rich repeats plete life cycle includes 41–47 d egg stage, 990–1,350 d larval stage, in the first domain and eight repeats in the second domain. Each 35–41 d pupal stage, and 3–8 d adult stage (Li et  al. 2011). The repeat had six cysteine residues, and each LDLa was followed by an reproductive success of all species, including insects, relies on Vg bio- EGF-like domain with/without a calcium-binding region. The EGF- synthesis and its uptake by VgR in the developing oocytes. As an like domain contained repeats of the LDLR YWTD motif. After the important host of traditional Chinese medicine caterpillar fungus, it second EGF-like domain, a TM domain spanning amino acids 1674– is of great significance to study the Vg and VgR in T. pui. 1691 and a cytoplasmic domain spanning amino acids 1692–1798 In this study, the full-length cDNA corresponding to the TpVg were predicted by the TMHMM server v. 2.0. and TpVgR genes was obtained, and the molecular characteristics of these two genes were also analyzed. The GL/ICG, DGXR, and K/ Phylogenetic Analyses of TpVg and TpVgR RXXR motifs, considered to be the conserved domains, have been A phylogenetic tree was constructed based on the distances of aa reported in many insect Vg proteins (Sappington and Raikhel 1998, sequences of TpVg and TpVgR. The phylogenetic tree showed Tufail and Takeda 2008, Upadhyay et  al. 2016). In earlier studies, that TpVg and Vgs of other Lepidoptera species clustered together the GL/ICG motif was assumed to be essential for the proper func- (Fig.  2). Vgs from the same insect order also clustered in the same tion of Vg during embryogenesis (Tufail et al. 2001). As is the case group, implying the conservation of Vgs and the closer relationships with other insects, a GLCG motif was found in the aa sequence within the same taxonomic status. A similar phylogenetic tree was of TpVg (Supplementary Fig.  2). Apart from the class  Insecta, constructed by comparing VgR aa sequences from different groups the GLCG motif was also present in Arachnoidea species, such of insects (Fig. 3). TpVgR and other VgRs of moth species formed a as Tetranychus urticae (Kawakami et  al. 2009), Panonychus citri Fig.  1. Conserved domain architecture of vitellogenin (TpVg) and vitellogenin receptor (TpVgR) in T.  pui. SP, signal peptide; LPD-N, lipoprotein N-terminal domain; DUF1943, domains of unknown function protein families; VWD, von Willebrand factor type D domain; LDLa, low-density lipoprotein receptor domain class A; EGF, epidermal growth factor-like domain; EGFC, calcium-binding EGF-like domain; LY, low-density lipoprotein receptor YWTD protein; EGFL, epidermal growth factor-like domain. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/23/4924664 by Ed 'DeepDyve' Gillespie user on 16 March 2018 4 Journal of Insect Science, 2018, Vol. 18, No. 2 Fig. 2. Phylogenetic analysis of vitellogenin in T. pui with that of insects from different orders. The phylogenetic tree was constructed by Mega 6 with a statistical method of neighbor joining. Numbers indicate bootstrap support values (%) based on 1,000 replicates. The numbers in the brackets indicate the GenBank accession for each insect. (Zhong et  al. 2015), and Ornithodoros moubata (Horigane et  al. four LDLa repeats in the first ligand-binding site and seven to eight 2010). A  DGXR motif is usually located 17–19 residues upstream in the second (Tufail and Takeda 2009, Lu et al. 2015). There were of the GL/ICG motif in most insect Vg sequences (Tufail and Takeda four LDLa repeats found in the first ligand-binding site of T.  pui, 2008), but that motif was not found in Rhyparobia maderae or in which is a typical feature in Lepidoptera species (Shu et al. 2011, Lin T.  pui in this study (Supplementary Fig.  2). In addition, some aa et al. 2013, Zhang et al. 2016). However, TpVgR bore eight LDLa residues, such as E (acidic), G, T and P (hydrophobic), and Y (hydro- repeats in the second ligand-binding site, which differs from other philic) were also highly conserved at the C-terminus of all the tested Vgs of Lepidoptera species; they generally have seven LDLa repeats. Vgs (Supplementary Fig. 3). However, the specific roles of these con- There are also eight LDLa repeats in the second ligand-binding site servative sites remain unclear. of the VgRs in other insects, including species of Hymenoptera, TpVgR also showed sequence similarities with other insect Coleoptera, Diptera, Hemiptera, Blattaria, which are also found in VgRs. The phylogenetic analysis indicated that TpVgR shares a Acarina species (Mitchell et al. 2007, Boldbaatar et al. 2010, Zhong homology with other Lepidopteran VgRs, such as Bombyx mori, et al. 2015). A.  selene, Helicoverpa armigera, Danaus plexippus, and Antheraea In some insect VgRs, the O-linked sugar domain (OLSD), which pernyi (Fig.  3). An analysis of the domain conservation of TpVgR is rich in S and/or T residue, is always between the last EGF pre- suggests that TpVgR in a member of the LDLR family and shares cursor domain and the TM domain (Tufail and Takeda 2007). several modular elements with other insect Vgs. TpVgR was shown No OLSD was found in TpVgR, which is consistent with VgRs of to contain the classical LBDs, EGF-like domains, and a TM domain. Solenopsis invicta, H.  armigera, and Drosophila melanogaster but TpVgR was found to have two ligand-binding sites that were char- not with B.  mori, A.  pernyi, and Nilaparvata lugens (Chen et  al. acterized by cysteine-rich LDLa repeats. Most insect Vgs have two to 2004, 2010; Lu et  al. 2015; Zhang et  al. 2016), implying that the Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/23/4924664 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 2 5 Fig. 3. Phylogenetic analysis of the vitellogenin receptor in T. pui with that of insects from different orders. The phylogenetic tree was constructed by Mega 6 with a statistical method of neighbor joining. Numbers indicate bootstrap support values (%) based on 1,000 replicates. The numbers in the brackets indicate the GenBank accession for each insect. Fig.  4. Relative expression of TpVg and TpVgR at different developmental stages. Samples include cDNA from third, fourth, fifth, sixth, seventh, and eighth instar larvae, 1-d-old pupae, 9-d-old pupae, 17-d-old pupae, 25-d-old pupae, 33-d-old pupae, and 1-d-old female adults. The mRNA levels were normalized using β-actin as a reference gene. Different letters indicate statistically significant differences at P < 0.05 (One-way ANOVA followed by LSD multiple comparison tests). The bars represent SD (n = 3). Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/23/4924664 by Ed 'DeepDyve' Gillespie user on 16 March 2018 6 Journal of Insect Science, 2018, Vol. 18, No. 2 Acknowledgments We would like to thank Cirentajie for the insect collection and rearing. This work was supported by the Special Fund for Agro-scientifc Research in the Public Interest (201403030) and the National Key Technology Research and Development Program of China (2011BAI13B06). The funders had no role in study design, data collection and analysis, decision to publish, or the prepara- tion of the manuscript. Conflict of interest These authors have no conflict of interest to declare. Supplementary Data Supplementary data are available at Journal of Insect Science online. References Cited Fig.  5. Relative expression of TpVg and TpVgR in the thorax and abdomen of T.  pui. The mRNA levels were normalized using β-actin as a reference Boldbaatar, D., R.  Umemiya-Shirafuji, M.  Liao, T.  Tanaka, X.  Xuan, and gene. Double asterisks indicate statistically significant differences at P < 0.01 K.  Fujisaki. 2010. Multiple vitellogenins from the Haemaphysalis lon- (Student’s t-test). The bars represent SD (n = 3). gicornis tick are crucial for ovarian development. J. 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Molecular Characterization and Expression of Vitellogenin and Vitellogenin Receptor of Thitarodes pui (Lepidoptera: Hepialidae), an Insect on the Tibetan Plateau

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Abstract

Vitellogenin (Vg) and vitellogenin receptor (VgR) play important roles in the vitellogenesis of insects. In this study, we cloned and characterized the two corresponding genes (TpVg and TpVgR) in an economically important insect, Thitarodes pui (Lepidoptera: Hepialidae), from the Tibetan plateau. The full length of TpVg is 5566 bp with a 5373 bp open reading frame (ORF) encoding 1,790 amino acids. Sequence alignment revealed that TpVg has three conserved domains: a Vitellogenin_N domain, a DUF1943 domain, and a von Willebrand factor type D domain (VWD). The full length of TpVgR is 5732 bp, with a 5397 bp ORF encoding 1798 amino acids. BLASTP showed that TpVgR belongs to the low-density lipoprotein receptor (LDLR) gene superfamily. Structural analysis revealed that TpVgR has a group of four structural domains: a ligand-binding domain (LBD), an epidermal growth factor (EGF)–precursor homology domain, a transmembrane (TM) domain, and a cytoplasmic domain. In addition, TpVgR has four cysteine-rich LDL repeats in the first ligand-binding site and seven in the second. Quantitative real-time polymerase chain reaction analysis revealed that the expression levels of TpVg and TpVgR are much higher in later pupa than in either the larval or adult stage, implying that the synthesis and uptake of Vg in T. pui occurs in the later pupal stage. These results will help us to understand the molecular mechanism of the reproductive capacity and will provide new insight into the mass rearing and utilization of T. pui. Key words: vitellogenin, vitellogenin receptor, Thitarodes pui Like other animals, insects require large amounts of vitellogenin (Vg) 1995, Cho and Raikhel 2001), Lepidoptera (Shu et  al. 2011, Qian to meet the nutritional needs of egg development. Vg, the precursor of et  al. 2015, Zhang et  al. 2016), Hemiptera (Upadhyay et  al. 2016), vitellin and the major yolk protein, is usually synthesized in the fat body, Coleoptera (Roy-Zokan et al. 2015), Hymenoptera (Chen et al. 2004), released into the hemolymph, and subsequently taken up by the vitel- and Blattaria (Tufail and Takeda 2007, 2008). The deduced amino logenin receptor (VgR) located on the external surface of competent acid (aa) sequences of these VgRs indicate that they are members of oocytes (Tufail and Takeda 2008, 2009). Vg has been studied exten- the low-density lipoprotein receptor (LDLR) family. In general, the sively in many insects of different orders. The identification of insect Vgs LDLR family members have five distinct, conserved motifs, including revealed that these Vgs belong to a large family of lipid transfer genes a ligand-binding domain (LBD) containing LDLR Class A  cysteine- and that they share similar structural motifs, including an N-terminal rich repeats, an epidermal growth factor (EGF)-like domain containing lipid binding domain (LPD_N), an unknown functional region LDLR Class B cysteine-rich repeats and YWXD repeats, an O-linked (DUF1943), and a von Willebrand factor type D similar domain (VWD) carbohydrate domain with a serine/threonine-rich track, a transmem- (Trewitt et al. 1992, Chen et al. 1994, Yano et al. 1994a, Veerana et al. brane (TM) domain, and a cytoplasmic tail (Tufail and Takeda 2007, 2014, Upadhyay et al. 2016, Zhao et al. 2016, Ibanez et al. 2017). 2009; Shu et al. 2011; Upadhyay et al. 2016; Zhang et al. 2016). VgR, the key element in the process of Vg uptake, plays an impor- Thitarodes pui (Lepidoptera: Hepialidae) is naturally dis- tant role during vitellogenesis. To date, the molecular characteristics tributed in Segrila Mountain of the Tibetan Plateau, which is of VgRs have been well documented in vertebrates and invertebrates characterized by a harsh climate with low temperatures and including many orders of insects, such as Diptera (Schonbaum et al. hypobaric hypoxia. T.  pui larvae are the hosts for the famous © The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/23/4924664 by Ed 'DeepDyve' Gillespie user on 16 March 2018 2 Journal of Insect Science, 2018, Vol. 18, No. 2 traditional Chinese medicinal caterpillar fungus, the ascomycete the pGEM-T vector (Takara, Dalian, China), and transformed into Ophiocordyceps sinensis (Berk.) Sung, Sung, Hywel-Jones, and Escherichia coli DH5α cells for amplification. Positive clones were Spatafora (syn. Cordyceps sinensis). assessed by PCR and sequenced. To obtain the full-length cDNA Once infection occurs, the fungus grows within the body cav- sequences of TpVg and TpVgR, a SMARTer RACE (rapid ampli- ity of the insect larvae and finally kills and mummifies them in fication of cDNA ends) cDNA Amplification Kit (Clontech) was their underground burrows. A  fruiting fungal body emerges from used according to the manufacturer’s instructions. The first round the front end of the caterpillar and protrudes from the ground in of RACE-PCR was performed using outer primer (Supplementary spring and early summer (Buenz et al. 2005). The medicinal activity Fig.  1 and Supplementary Table  1) and a universal primer mix is derived from the parasite complex of the mummified caterpillar (UPM) with the following protocol: one cycle of predenaturing at and the fungal stromata (Zhang et al. 2009). 94°C for 5 min,; 35 cycles of 30 s at 95°C, 30 s at 65°C, and 2 min The complete life cycle of T.  pui lasts at least 3 yr under natu- at 72°C; and a final extension at 72°C for 5 min. The second round ral conditions (Li et al. 2011). A better understanding of the molec- of RACE-PCR was performed using inner primers (Supplementary ular mechanisms regulating reproduction can indicate potential Fig.  1 and Supplementary Table  1) and a nested universal primer approaches for the mass rearing of these economically important (NUP). The second RACE-PCR protocol was performed as follows: insects. Vg and VgR are important molecular markers for insect one cycle of predenaturing at 94°C for 5 min; 35 cycles of 30 s at fecundity. In this study, we cloned the full lengths of vitellogenin 95°C, 30 s at 68°C, and 2 min at 72°C; and a final extension at 72°C (TpVg) and the vitellogenin receptor (TpVgR) of T.  pui, character- for 5 min. The RACE products were gel purified and sequenced as ized the molecular structure of the corresponding deduced proteins, described above. and analyzed their gene expression profiles at different developmen- tal stages. Our results are expected to provide the theoretical basis Sequence Comparisons and Phylogenetic Analysis for the conservation and utilization of this economically important The sequence similarities were analyzed using the online BLASP insect. program on the NCBI website (https://blast.ncbi.nlm.nih.gov/). The open reading frames (ORFs) of TpVg and TpVgR were pre- dicted using the NCBI ORF finder (https://www.ncbi.nlm.nih.gov/ Materials and Methods orffinder/). The molecular weights and isoelectric points (pIs) of Insect Rearing and Collection the deduced protein sequences were predicted using the ExPASy T.  pui samples were collected in the Tibetan Plateau Peculiar Bio- proteomics server (http://www.expasy.org). The domain architec- resources Research Station of Sun Yat-sen University at Nyingchi in ture and the conserved domains were analyzed using the online the Tibet Autonomous Region (4,156 m altitude, 29°36′N, 94°36′E) servers of Scan-Prosite (http://prosite.expasy.org/scanprosite/), from June to August in 2014. The larvae were reared as previously SMART (http://smart.embl-heidelberg.de/), and InterProScan described (Sun et al. 2011, Wu et al. 2015). The larvae of different (http://ebi.ac.uk/Tools/pfa/iprscan/). The signal peptide was pre- instars (third through eighth), female pupae of different ages (1, 9, dicted by using the SignalP 4.1 Server (http://www.cbs.dtu.dk/ 17, and 33 d old), and 1-d-old adult females were used for the ana- services/SignalP). The potential O-linked glycosylation sites were lysis of gene expression. All of the samples at different developmen- evaluated using the GPP Prediction Server (http://comp.chem.not- tal stages were freshly frozen in a Sample Protector (Takara, Tokyo, tingham.ac.uk/glyco/), and the TM regions were predicted using Japan) at −80°C until RNA isolation. the TMHMM server v2.0 (http://cbs.dtu.dk/services/TMHMM/). All the computational programs were run with the recommended default values set up by the programs. The phylogenetic tree was RNA Isolation and cDNA Synthesis constructed with MEGA 6.0 by using the neighbor-joining method Total RNA was extracted from the whole body of T.  pui using with a bootstrap of 1,000 replicates. TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions, and RNA purity and degradation were checked on 1% agarose gels and Bioanalyzer 2100 system (Agilent Technologies). Developmental Expression Profile of TpVg and For tissue-specific expression profiles, total RNAs were extracted TpVgR from the thorax and abdomen parts of newly emerged female adults Equal quantities of cDNA (1  µg) from different developmental which were stored separately in a refrigerator at −80°C. The RNA stages were used for a relative expression analysis of TpVg and sample was dissolved in 20 µl diethylpyrocarbonate (DEPC)-treated TpVgR messenger RNA (mRNA) using a real-time PCR machine H O and assessed by spectrophotometry (Nanodrop 2000, Thermo (LightCycler 480 real-time PCR machine, Roche Applied Science, Fisher Scientific, DE). A PrimeScript Reagent Kit with gDNA Eraser Switzerland) with SYBR Green (Invitrogen). β-actin was used as an (TaKaRa, Tokyo, Japan) was used to synthesize first-strand comple- internal control based on a previous study (Sun et al. 2011). Primers mentary DNA (cDNA) with 1µg total RNA in a 20 µl reaction mix- used for quantitative PCR (qPCR) are listed in Supplementary ture following the manufacturer’s recommendations. Table 1. The qPCR amplifications were conducted in a total volume of 10 µl, including 1 µg of cDNA, 5 µl of SYBR Green I Master mix, Molecular Cloning of TpVg and TpVgR 0.5  µl of each primer (10  µM), and a certain amount of ddH O. The qPCR program included one cycle of 95°C for 10 s, followed Two partial sequences of both Vg and VgR were identified from by 40 cycles of 95°C for 5 s, 60°C for 20 s, and 72°C for 20 s. To the pupal transcriptome of T. pui (unpublished data). The fragment ensure reliability, each sample included three biological replicates. sequences of TpVg and TpVgR were further verified by polymer - The melt curve genotyping analysis was performed to confirm the ase chain reaction (PCR) amplification using the primers listed in homogeneity of the PCR products. Reactions were performed in Supplementary Table  1. The PCR program was performed as fol- triplicate for each sample, and gene expression levels were normal- lows: one cycle of predenaturing at 95°C for 5 min; 32 cycles of 95°C −∆∆ ized against T.  pui β-actin according to the 2 CT method (Livak for 30 s, 53°C for 30 s, and 72°C for 2 min; and then 72°C for 5 min and Schmittgen 2001). for elongation. The PCR products were then gel purified, cloned into Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/23/4924664 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 2 3 cluster. Other VgRs from the same insect orders were also clustered. Results In the phylogenetic tree of VgRs, Lepidoptera VgRs formed a single Sequence and Structural Analysis of TpVg and branch and was separated from other species (Fig. 3). However, the TpVgR dendrogram of Vgs showed that the Vgs of Diptera and Lepidoptera The full sequence of TpVg was 5566  bp, including an 83  bp 5′ species were close together (Fig. 2), suggesting that the Vgs of those untranslated region (UTR), a 5373 bp ORF encoding 1,790 aa res- two insect orders share a closer ancestry than other insects. idues, and a 110  bp 3′ UTR (GenBank accession no. MF622538). The theoretical molecular weight was 204.43 kDa, and the pI was Expression Profile of TpVg and TpVgR 6.51. A BLASTP analysis showed that the aa sequence of TpVg had a The expression analysis of TpVg and TpVgR was conducted during 30 to 40% similarity with other insects from different orders. TpVg the larval, pupal, and adult stages by real-time PCR (Fig.  4). The showed a 34 % and 33 % similarity with Operophtera brumata and expression level of TpVg was normalized with a reference gene, Actias selene, respectively. As with other insects, TpVg in T. pui also β-actin of T.  pui. The results showed that TpVg had no detectable contained the typical conserved domains of the Vg protein. expression in the larval stage, but it had a high expression during Domain architecture analysis by SMART showed that the TpVg the pupae stage, decreasing significantly in adult females (Fig.  4). aa sequence has three conserved domains (Fig. 1): the vitellogenin_N The expression of TpVg began to increase in the pupal stage and domain, the DUF1943 domain, and the VWD. A signal peptide with reached its peak in the 25 and 33-d-old pupae, i.e., the final stage 18 amino acids (MRTFVLFALLATAFCANS) was found in the ana- of pupae. Then, the expression level of TpVg decreased significantly lysis of the predicted aa sequence of TpVg. In addition, the deduced in the adult stage. TpVgR had an expression profile similar to that aa sequences of TpVg included four N-glycosylation sites (NXS/T) of TpVg. TpVgR was found to have lower expression in the early and seven K/RXXR cleavage sites. pupal stage and then a remarkably high expression in the 17-d-old The full sequence of TpVgR was 5732  bp, including a 74  bp pupae, subsequently reaching a maximum expression level in the 5′ UTR, a 5397 bp ORF encoding 1,798 aa residues, and a 261 bp later pupae stage. Then, a lower expression level was maintained 3′ UTR (GenBank accession no. MF622539). The theoretical molec- in the adult period. In addition, the tissue-specific expression profile ular weight was 200.47 kDa, and the pI was 5.46. The analysis of the revealed that TpVg and TpVgR were highly expressed in the abdo- predicted aa sequence revealed that a signal peptide with 27 amino men which contained the reproductive organ-ovary (Fig. 5). acids (MGHADLLHIALIIVVNVFLLKWKMAGA) was located at the N-terminal of TpVgR. The protein sequence analysis indicated that TpVgR contained Discussion the characteristic features of the LDLR family (Fig.  1). TpVgR T. pui is a holometabolous insect with a long life history. The com- showed two LBDs with four class A  (LDLa) cysteine-rich repeats plete life cycle includes 41–47 d egg stage, 990–1,350 d larval stage, in the first domain and eight repeats in the second domain. Each 35–41 d pupal stage, and 3–8 d adult stage (Li et  al. 2011). The repeat had six cysteine residues, and each LDLa was followed by an reproductive success of all species, including insects, relies on Vg bio- EGF-like domain with/without a calcium-binding region. The EGF- synthesis and its uptake by VgR in the developing oocytes. As an like domain contained repeats of the LDLR YWTD motif. After the important host of traditional Chinese medicine caterpillar fungus, it second EGF-like domain, a TM domain spanning amino acids 1674– is of great significance to study the Vg and VgR in T. pui. 1691 and a cytoplasmic domain spanning amino acids 1692–1798 In this study, the full-length cDNA corresponding to the TpVg were predicted by the TMHMM server v. 2.0. and TpVgR genes was obtained, and the molecular characteristics of these two genes were also analyzed. The GL/ICG, DGXR, and K/ Phylogenetic Analyses of TpVg and TpVgR RXXR motifs, considered to be the conserved domains, have been A phylogenetic tree was constructed based on the distances of aa reported in many insect Vg proteins (Sappington and Raikhel 1998, sequences of TpVg and TpVgR. The phylogenetic tree showed Tufail and Takeda 2008, Upadhyay et  al. 2016). In earlier studies, that TpVg and Vgs of other Lepidoptera species clustered together the GL/ICG motif was assumed to be essential for the proper func- (Fig.  2). Vgs from the same insect order also clustered in the same tion of Vg during embryogenesis (Tufail et al. 2001). As is the case group, implying the conservation of Vgs and the closer relationships with other insects, a GLCG motif was found in the aa sequence within the same taxonomic status. A similar phylogenetic tree was of TpVg (Supplementary Fig.  2). Apart from the class  Insecta, constructed by comparing VgR aa sequences from different groups the GLCG motif was also present in Arachnoidea species, such of insects (Fig. 3). TpVgR and other VgRs of moth species formed a as Tetranychus urticae (Kawakami et  al. 2009), Panonychus citri Fig.  1. Conserved domain architecture of vitellogenin (TpVg) and vitellogenin receptor (TpVgR) in T.  pui. SP, signal peptide; LPD-N, lipoprotein N-terminal domain; DUF1943, domains of unknown function protein families; VWD, von Willebrand factor type D domain; LDLa, low-density lipoprotein receptor domain class A; EGF, epidermal growth factor-like domain; EGFC, calcium-binding EGF-like domain; LY, low-density lipoprotein receptor YWTD protein; EGFL, epidermal growth factor-like domain. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/23/4924664 by Ed 'DeepDyve' Gillespie user on 16 March 2018 4 Journal of Insect Science, 2018, Vol. 18, No. 2 Fig. 2. Phylogenetic analysis of vitellogenin in T. pui with that of insects from different orders. The phylogenetic tree was constructed by Mega 6 with a statistical method of neighbor joining. Numbers indicate bootstrap support values (%) based on 1,000 replicates. The numbers in the brackets indicate the GenBank accession for each insect. (Zhong et  al. 2015), and Ornithodoros moubata (Horigane et  al. four LDLa repeats in the first ligand-binding site and seven to eight 2010). A  DGXR motif is usually located 17–19 residues upstream in the second (Tufail and Takeda 2009, Lu et al. 2015). There were of the GL/ICG motif in most insect Vg sequences (Tufail and Takeda four LDLa repeats found in the first ligand-binding site of T.  pui, 2008), but that motif was not found in Rhyparobia maderae or in which is a typical feature in Lepidoptera species (Shu et al. 2011, Lin T.  pui in this study (Supplementary Fig.  2). In addition, some aa et al. 2013, Zhang et al. 2016). However, TpVgR bore eight LDLa residues, such as E (acidic), G, T and P (hydrophobic), and Y (hydro- repeats in the second ligand-binding site, which differs from other philic) were also highly conserved at the C-terminus of all the tested Vgs of Lepidoptera species; they generally have seven LDLa repeats. Vgs (Supplementary Fig. 3). However, the specific roles of these con- There are also eight LDLa repeats in the second ligand-binding site servative sites remain unclear. of the VgRs in other insects, including species of Hymenoptera, TpVgR also showed sequence similarities with other insect Coleoptera, Diptera, Hemiptera, Blattaria, which are also found in VgRs. The phylogenetic analysis indicated that TpVgR shares a Acarina species (Mitchell et al. 2007, Boldbaatar et al. 2010, Zhong homology with other Lepidopteran VgRs, such as Bombyx mori, et al. 2015). A.  selene, Helicoverpa armigera, Danaus plexippus, and Antheraea In some insect VgRs, the O-linked sugar domain (OLSD), which pernyi (Fig.  3). An analysis of the domain conservation of TpVgR is rich in S and/or T residue, is always between the last EGF pre- suggests that TpVgR in a member of the LDLR family and shares cursor domain and the TM domain (Tufail and Takeda 2007). several modular elements with other insect Vgs. TpVgR was shown No OLSD was found in TpVgR, which is consistent with VgRs of to contain the classical LBDs, EGF-like domains, and a TM domain. Solenopsis invicta, H.  armigera, and Drosophila melanogaster but TpVgR was found to have two ligand-binding sites that were char- not with B.  mori, A.  pernyi, and Nilaparvata lugens (Chen et  al. acterized by cysteine-rich LDLa repeats. Most insect Vgs have two to 2004, 2010; Lu et  al. 2015; Zhang et  al. 2016), implying that the Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/23/4924664 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 2 5 Fig. 3. Phylogenetic analysis of the vitellogenin receptor in T. pui with that of insects from different orders. The phylogenetic tree was constructed by Mega 6 with a statistical method of neighbor joining. Numbers indicate bootstrap support values (%) based on 1,000 replicates. The numbers in the brackets indicate the GenBank accession for each insect. Fig.  4. Relative expression of TpVg and TpVgR at different developmental stages. Samples include cDNA from third, fourth, fifth, sixth, seventh, and eighth instar larvae, 1-d-old pupae, 9-d-old pupae, 17-d-old pupae, 25-d-old pupae, 33-d-old pupae, and 1-d-old female adults. The mRNA levels were normalized using β-actin as a reference gene. Different letters indicate statistically significant differences at P < 0.05 (One-way ANOVA followed by LSD multiple comparison tests). The bars represent SD (n = 3). Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/23/4924664 by Ed 'DeepDyve' Gillespie user on 16 March 2018 6 Journal of Insect Science, 2018, Vol. 18, No. 2 Acknowledgments We would like to thank Cirentajie for the insect collection and rearing. This work was supported by the Special Fund for Agro-scientifc Research in the Public Interest (201403030) and the National Key Technology Research and Development Program of China (2011BAI13B06). The funders had no role in study design, data collection and analysis, decision to publish, or the prepara- tion of the manuscript. Conflict of interest These authors have no conflict of interest to declare. Supplementary Data Supplementary data are available at Journal of Insect Science online. References Cited Fig.  5. Relative expression of TpVg and TpVgR in the thorax and abdomen of T.  pui. The mRNA levels were normalized using β-actin as a reference Boldbaatar, D., R.  Umemiya-Shirafuji, M.  Liao, T.  Tanaka, X.  Xuan, and gene. Double asterisks indicate statistically significant differences at P < 0.01 K.  Fujisaki. 2010. Multiple vitellogenins from the Haemaphysalis lon- (Student’s t-test). The bars represent SD (n = 3). gicornis tick are crucial for ovarian development. J. 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