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Cloning and characterization of hexamerin in Spodoptera exigua and the expression response to insecticide exposure
Accepted Manuscript Cloning and characterization of hexamerin in Spodoptera exigua and the expression response to insecticide exposure
Shiyu Wang, Bo Hu, Qi Wei, Jianya Su PII: DOI: Reference:
S1226-8615(19)30001-9 https://doi.org/10.1016/j.aspen.2019.04.006 ASPEN 1375
To appear in:
Journal of Asia-Pacific Entomology
Received date: Revised date: Accepted date:
1 January 2019 15 March 2019 16 April 2019
Please cite this article as: S. Wang, B. Hu, Q. Wei, et al., Cloning and characterization of hexamerin in Spodoptera exigua and the expression response to insecticide exposure, Journal of Asia-Pacific Entomology, https://doi.org/10.1016/j.aspen.2019.04.006
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ACCEPTED MANUSCRIPT Cloning and characterization of hexamerin in Spodoptera exigua and the expression response to insecticide exposure Shiyu Wang, Bo Hu, Qi Wei, and Jianya Su* Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Nanjing 210095,
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China.
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* Corresponding authors:
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Dr. Jianya Su Department of Pesticide Science,
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College of Plant Protection,
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Nanjing Agricultural University, Nanjing 210095, China.
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Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract Hexamerins are hemolymph-proteins, which are mainly considered as storage proteins for non-feeding stages, and also undertake other roles during insect development and growth, however the characterization of hexamerin proteins in Spodoptera exigua is less understood. In this study five new hexamerin genes were identified and a total seven hexamerin genes were reported in S. exigua. These hexamerins contain the typical domains of hemocyanin at
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the N-terminal, C-terminal and in the middle of their protein sequences. These genes are mainly expressed in fat
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body, and the signal peptide sequences at their N-terminal of protein sequences can drive the expressed protein to
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excrete into hemolymph after synthesis. The phylogenetic analysis and amine acid composition revealed S. exigua express five different types of hexamerins: 1) Storage protein rich in methionine residue (MRSP), 2) Storage protein
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moderately rich in methionine (MMRSP), 3) Hexamerin with high composition of aromatic amino acids (Arylphorin), 4)
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Arylphorin-like hexamerin, and 5) Riboflavin-binding hexamerin (RbH). The phylogenetic pattern combined with the comparison of conserved histidine residues in copper binding sites of hexamerins revealed basal position of RbH and
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the evolutionary pathway in lepidopteran hexamerins. Finally, the induction expression of hexamerins by insecticide,
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lambda-cyhalothrin, were analyzed, results showed that lambda-cyhalothrin exposure may down-regulate their expression. This study increased the gene number of hexamerin to seven, and reported their expression and
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structural characterizations, the finding will facilitate the understand of hexamerin in other insects.
Key words: Hexamerin, Spodoptera exigua, phylogenetic analysis, insecticide induction, lambda-cyhalothrin.
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ACCEPTED MANUSCRIPT 1. Introduction Hexamerins belong to an arthropod protein superfamily that additionally includes hemocyanins, phenoloxidases and hexamerin receptors (Burmester, 2002). There were different nomenclatures for hexamerins according to occurrence in insect species (calliphorin, manduci, etc), their particular amino acid composition (arylphorin, methionine-rich storage protein), or simply referred as “(larval) storage proteins”. The name “hexamerin” refers to
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their composition of six homo- or hetero-hexamer subunits ranging 70 - 90 kDa to form a native molecular mass of
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about 500 kDa (Burmester and Scheller, 1999; Telfer and Kunkel, 1991). Hexamerins resemble the arthropod
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hemocyanins in terms of structure and sequence (Burmester and Schellen, 1996). While hemocyanins serve as respiratory proteins, hexamerins do not bind copper and thus oxygen. Molecular phylogenetic analyses have shown
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that hexamerins evolved within the hexapod lineage, after the divergence from hemocyanin of crustaceans (Pick et
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al., 2009).
Hexamerins have been discovered in all insect species studied so far and are mainly expressed by fat body and
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exported into the hemolymphs (Burmester, 2004). Initially, they have been considered solely as storage proteins that
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provide amino acids and energy for non-feeding periods such as molting process. This view is supported by their massive accumulation during late nymph and larval stages, where they may account for up to 50% of the soluble
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proteins. Hexamerins disappear during metamorphosis (Telfer and Kunkel, 1991) and hexamerin-derived amino acid
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are incorporated into adult tissues (Pan and Telfer, 2001). Analogous to the function of vertebrate serum proteins, hexamerins may transport hormones such as ecdysteroids and juvenile hormone (Telfer and Kunkel, 1991). The regulation of juvenile hormone level by hexamerins may also be instrumental in caste differentiation of termites (Zhou et al., 2007). There is also evidence that at least some hexamerins are involved in immune response to bacteria (Eliautout et al., 2016; Ma et al., 2012; Poopathi et al., 2014), Plasmodium (Lombardo and Christophides, 2016), parasitoid (Zhu et al., 2009), and acclimation (Sonoda et al., 2006). Hexamerins may also bind to other organic compounds such as riboflavin (Magee et al., 1994; Miller and Silhacek, 1995; Pan and Telfer, 1999) or certain 3
ACCEPTED MANUSCRIPT insecticides (Haunerland and Bowers, 1986b). Hexamerin was found to be inducible by Cry1Ac toxin in Heliceverpa armigera, and the binding of hexamerin with toxin leads to the formation of insoluble aggregates, which may hamper the toxicity of Bt toxins (Ma et al., 2005). An 80-kDa protein, which was idenfied as hexamerin, was found to be highly expressed in Culex quinquefasciatus resistant to toxin of Bacillus sphaericus, and. binding of toxins with this protein possibly lead to the toxin sequestration, however, the role of hexamerin expression in toxin resistance
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remains elusive (Poopathi et al., 2014).
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Spodoptera exigua is an important insect pest on vegetable crops, the frequent applications of insecticides have
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resulted in rapid development of insecticide resistance in the field populations of this pest from worldwide (Su and Sun, 2014). In present research the genes for hexamerins in S. exigua were cloned and analyzed for the expression
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level in different tissues of larvae. We obtained seven hexamerin cDNA sequences which encode proteins with typical
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of hexamerin domains. The expression changes of six hexamerin genes in larvae under insecticide challenge were analyzed and the function and phylogenetic of hexamerins was also discussed.
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2. Materials and methods
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2.1 Insects
A laboratory strain of S. exigua, which was obtained from Wuhan Kernel Biopesticide Company, Wuhan, China,
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in May 2001, was used in this study. This strain was maintained in laboratory without exposure to any insecticide. All
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stages of insects were maintained in an insectary at 27 ± 1°C under a photoperiod of 16:8 (L:D) and 50 % - 70 % RH. The larvae were reared with artificial diet and adults were fed 10 % honey solution (Lai and Su, 2011) 2.2 RNA extractions and cDNA synthesis For gene cloning the third instar larvae of S. exigua were collected, and total RNAs were extracted using TRIZOL regent (Invitrogen. USA) following the manufacturer’s instructions. The quality of total RNA was check by agarose gel electrophoresis and the quantity of total RNA was determined with NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). First-strand cDNA was synthesized using Supermo III RT Kit (Bio Teke, China). 4
ACCEPTED MANUSCRIPT 2.3 Cloning and sequence analysis of hexamerins According to the transcriptome data of S. exigua (Li et al., 2013; Pascual et al., 2012) the sequences of seven hexamerin genes were verified by gene clone and sequencing. The gene-specific primers were designed to directly amplify the nucleotide sequence for each gene’s ORF (Table S1). PCRs were made using Taq Master Mix (Vazyme Biotech Co., Ltd. Nanjing, China) according to following PCR protocol: 1 cycle of denaturation at 95 °C for 3 min, 35
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amplification cycles (94 °C, 50 s; 50 °C, 50 s; 72 °C, 1 min), and finally extention at 72 °C for 10 min. The PCR
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reactions were made in 50 μL system containing PCR reagents according to the instructions. PCR products were
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electrophoresed on 1.5 % agarose gel, extracted by gel extraction kit (E.Z.N.ATM Gel Extration Kit) and were subcloned into pMD19-T vector (Takara, Dalian, China). The positive clones were sent for sequencing by GENEWIZ
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Biological technology CO., Ltd (Suzhou, China).
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Sequence alignment was made using Clustal X6.0 software. Signal peptides were predicted using the web service of http://www.cbs.dtu.dk/services/SignalP/, and the CD search tool of NCBI was used to predict the structural
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domains of proteins. The maximum likelihood phylogenetic tree (ML tree) of hexamerins was constructed using
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amino acid sequences of hexamerins from S. exigua and other insect orders by MEGA6.0. Bootstrap analysis was performed using 1000 replicates to evaluate the significance of the nodes (Tamura et al., 2013).
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2.4 Quantitative real-time PCR (qRT-PCR)
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For analysis pattern of hexamerin genes, hemocytes of 1-day-old 5th instar larvae were collected and their head, fat body, midgut, and epidermis were dissected according to previous protocols (Hu et al., 2019; Kim and Kim, 2010; Tian et al., 2018). Total RNAs were isolated using TRIZOL regent and subjected to DNase I to remove any residual genomic DNA according to the manufacturer’s instructions. The isolated RNAs were reverse transcribed using the HiScriptTM Q Select RT SuperMix (Vazyme, Nanjing, China). The mRNA abundance of hexamerin genes was estimated by qRT-PCR in an ABI 7300 Fast Real-Time PCR System (Applied Biosytems, Foster City, CA) using AceQ qPCR SYBR Green Master Mix kit (Vazyme, China) according to the instruction. GAPDH was used as internal reference gene for 5
ACCEPTED MANUSCRIPT analyses (Zhu et al., 2014). The PCR primers for hexamerins were designed using the Primer 5 software. The specificity and sensitivity of primers for qRT-PCR assays were evaluated through melting curve analysis coupled with agarose gel electrophoresis (distinct single peaks in melting curve analysis, unique clear PCR product in agarose gel electrophoresis). PCR efficiencies were calculated from the standard curve (Table S2). A reverse transcription negative control (without reverse transcriptase) and a non-template negative control were included for each primer
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set to confirm the absence of genomic DNA and to check for primer-dimer or contamination in the reactions,
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respectively. The PCR system contained 0.8 µL of the forward and reverse primers (10 mmol/L), 10 µL 2 x SYBR Green
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Master Mix, 0.4 µL ROX X Reference Dye2, 1 µL of the diluted cDNA samples and nuclease-free water in a final volume of 20 µL. The quantification analysis for each gene was made in three biological samples (n = 3) and every
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biological sample was assayed with three mechanical repeats. The reaction conditions were as follows: an initial
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denaturation step of 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60 °C for 40 s. Melt curve analysis of the products was as follows: heating to 95°C for 15 s, decrease to 60°C for 60s and then 95°C for 15 s. Relative
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expression levels of these genes were calculated by the 2-∆∆T method (Livak and Schmittgen, 2001). All method and
guidelines (Bustin et al., 2009).
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2.5 Insecticide exposure
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data were confirmed to follow the minimum information for publication of quantitative real time PCR experiments
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In the research of gene expression responses in insects exposed to insecticides a sublethal concentration of insecticide was applied to challenge insects to avoid the high mortality at high concentration of insecticide. For insecticide induction studies in this study the 3rd instar larvae of S. exigua were exposed to LC20 concentration (0.25mg/L) of lambda-cyhalothrin (Jiangsu Yangnong Chemical Company, China) and collected after insecticide exposure for 24 h and 48 h. The total RNAs were isolated as previous protocol. Forty larvae were used as one treatment, and three replicates were applied in insecticide exposure experiment, therefore, totally 120 larvae were exposed to insecticide, and then among the alive larvae half were harvested at 24 h, and the remaining alive larvae 6
ACCEPTED MANUSCRIPT at 48 h for RNA isolation. The total RNA isolated from ten individual larvae were pooled as one biological sample for quantitative real-time PCR according to the procedure described in 2.4 section.
3. Results 3.1 The identification and characterization of hexamerins from S. exigua Seven hexamerin genes were identified from transcriptome sequences and further confirmed through RT-PCR
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and sequencing from larvae of S. exigua, the structure information including theoretical isoelectric point and the
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theoretical molecular weight of proteins coding by six sequences with complete open read frame (ORF) are listed in
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Table 1. The seventh one is a partial sequence lacking ORF, therefore its structure information is not listed. The six hexamerin genes encode proteins with 691- 753 amino acids; each protein contains signal peptide of 16 - 19 amino
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acids at N-terminal. Three conserved hemocyanin domains are found at the N-terminal, C-terminal of sequences and
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in the middle of sequences, respectively (Figure 1), suggesting they belong to hemocyanin family protein. Different from hemocyanins, hexamerin proteins lost most of the copper binding sites in sequences, we compare the copper
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binding sites of hexamerins from S. exigua with that from other lepidoptera insects, springtail, dipluran and crustean
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(Figure S1). The hemocyanins of lobsters in Crustean and springtails in Collembola keep these six conserved histidine (His) residues for copper binding sites, Dipluran lost four of the six, SeRbH of S. exigua also lost four of the six His
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residues, however, only one His residue are conserved in other six hexamerins from S. exigua (Figure S1).
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Hexamerins from S. exigua have different composition of amino acids, protein encoded by KY419212 contains relatively high content of methionine (8.26 %), therefore belongs to methionine-rich soluble protein group (MRSP). Proteins encoded by KY419215 and KY419213 contains moderate content of methionine, 4.78 % and 4.14 % respectively, hence belongs to the moderately methionine-rich soluble protein group (MMRSP). KY419214 and KY419217 encode protein with high composition of aromatic amino acids (phenylanine and tyrosine), 19.10 % and 18.53 %, respectively (arylphorin, ARYL). KY419216 encodes protein (RbH) with very low composition of methionine (1.56 %) (Figure 2). The average content of methionine and aromatic amine acids were also analyzed for the five 7
ACCEPTED MANUSCRIPT types of hexamerins from lepidopteran insects and compared with that of hemocyanin from Crustaea and Collembora (Figure 3). The average percentage of methionine is 1.70 %, 2.4 % and 2.25 % in RbH, ARYL and ARYL-like hexamerins, respectively, which is similar with that of lobsters (2.28 %) and springtails (2.21 %). However, there is a significant increase of methionine content in MMRSP (4.92 %), and a further obvious increase in MRSP (7.94 %) (Figure 3A). The mean content of aromatic amine acids in RbH is 10.62 %, it is also similar with that of lobsters
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(9.78 %) and springtails (8.76 %). Their content is increased significantly in ARYL-like hexamerin (13.79 %), and a
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further significant increase is observed in ARYL (18.39 %). However, it is decreased in MMRSP significantly (11.5 %),
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and then slightly lowered down to 9.62 % in MRSP (Figure 3B). 3.2 The phylogenetic analysis of hexamerins
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To understand the evolution of hexamerins we collected the hexamerin sequences of insects available from the
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NCBI database. Phylogenetic tree reconstructions were carried out using maximum likelihood method based on the amino acid sequences. No hexamerin is found in the available sequences of Crustacea, and hexamerins appear in the
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order of Collembora, which is positioned at the base of hexamerin tree, and hemocyanin and hexamerin coexist in
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this order. The hexamerins from Hemiptera are close to that of Apteygota. Hexamerins from holometabolous insects form single clades in each order except Lepidoptera. In lepidopteran insects hexamerins are split into two diverge
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that of Diptera (Figure 4).
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clades, one small branch (RbH) and big branch with other four types of hexamerins. The big branch is parallel with
Based on the ML tree and amino acid composition the hexamerins from lepidoptera insects are divided into five groups: MRSP protein belongs to the methionine-rich hexamerins group; MMRSPα and MMRSPβ belong to the moderately methionine-rich hexamerins group; ARYLα and ARYLβ belong to the arylphorins group; RbH belong to the riboflavin-binding hexamerins group and an ARYL-like group. RbH is positioned at the root of lepidopteran hexamerin clade, and then the ARYL-like hexamerin, which was first predicted from Manduca sexta by Burmester in 2015. This sequence was also identified in S. exigua. And further evolution give rise to ARYL and MMRSP/MRSP hexamerins 8
ACCEPTED MANUSCRIPT (Figure 4). 3.3 Expression pattern of hexamerins in S. exigua larvae In the first stage of the study we find only six hexamerin gene in S. exigua, the arylphorine-like hexamerin was searched out when we blasted insect hexamerins in the NCBI database to construct the phylogenetic tree, therefore the expression profiles of only six hexamerin genes were determined in the larvae of beet armyworm by qRT-PCR.
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The relative expression level of mRNAs was measured for each of them in the head, fat body, midgut, epidermis and
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hemocytes of the 1-day-old 5th instar larvae. The results show that the expressions are detected in all the analyzed tissues for six hexamerins, however, each hexamerin has specific expression pattern in different tissues of larvae
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(Figure 5). SeMRSP has higher expression level in fat body and epidermis than in other tissues. For SeMMRSP the
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highest expression level is observed in fat body, and then in hemocytes, and low level of expression is observed in
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head, epidermis and midgut. For SeMMRSP the highest expression is found in fat body and hemocytes, and very low expression in head, epidermis and midgut. The rank of expression levels for SeAYRL is that: fat body > head >
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epidermis, hemocyte > midgut. The ranking for SeAYRL is fat body > epidermis > hemocyte, head, and midgut. Fat
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body and hemocytes have higher expression of SeRbH than other tissues, and midgut has the lowest expression of this gene. The common characteristic for expression is that six hexamerins are highly expressed in fat body, however
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exigua.
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very low in midgut of larvae. This result demonstrates that fat body is the main expression tissue for hexamerin in S.
3.4 Expression response of hexamerins to insecticide challenges To understand the responses of hexamerin expression in S. exigua challenged by insecticide qRT-PCR were performed to determine the changes in transcriptional level for each of the hexamerin genes. Lambda-cyhalohrin exposure at the LC20 concentration (0.25 mg/L) down-regulates the expression of these hexamerins. The expression levels of SeMRSP, SeMMRSP and SeARYL are significantly reduced at 24h and 48h exposure post. SeARYL is down-regulated at 48h exposure post. The expressions of SeRbH and SeMMRSP are slightly reduced at 24h, and then 9
ACCEPTED MANUSCRIPT recover to the control level at 48h (Figure 6). The expressions of hexamerin genes in larvae of S. exigua are influenced by the insecticide challenge.
4. Discussion 4.1 The phylogenetic analysis of hexamerins within insects In a previous report two hexamerin genes (SeHex and SeSP1) were cloned and characterized from S. exigua
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(Tang et al., 2010), in this study we identified other five hexamerins, which were not reported previously in this
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species, totally seven hexamerin genes of S. exigua were identified. Before our study only six hexamerin genes had
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been identified in Manduca sexta, and were classified into five distinct groups (Burmester, 2015). The hexamerins from S. exigua are also belonging to these five groups based on amino acid composition and evolutionary analysis
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(Figure 4).
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Hexamerins have recently been applied in the phylogenetic analysis of insect orders, and the trees constructed from insect hexamerins are consistent with the generally accepted relationships of insect taxa, however, these
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analysises were mainly made on hexamerins from “lower” insects (Burmester, 2002; Burmester, 2004; Burmester et
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al., 1998; Hagner-Holler et al., 2007; Pick et al., 2009; Xie and Luan, 2014; Zhang et al., 2017). Here we incorporated seven hexamerin sequences of S. exigua into the phylogenetic analysis and exhibited an evolutionary pattern
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different from previous one (Burmester, 2015; Burmester et al., 1998). The ML tree demonstrates the complex
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evolution of hexamerins in Lepidoptera, which exceeds the diversity of other insect orders. The diversity may be partly due to an insufficient understanding on hexamerins in other insect taxa, or the biased sequencing approaches (Burmester et al., 1998).
RbH is positioned at the base of lepidopteran clade of hexamerins, and the content of methionine or aromatic amine acids in RbH is much similar with that of hemocyanin from Crustaea and Collembora, mightly suggesting RbH is the archetype of hexamerin in Lepidoptera. Different from other insect orders, lepidopteran evolution gave rise to the distinct aromatic or methionine-rich hexamerins to cope with undefined requirement during metamorphosis in 10
ACCEPTED MANUSCRIPT this order. Arylphorin was firstly identified in Hyalophora cecropia (Lepidoptera) and named due to the high content of aromatic amino acids (Telfer et al., 1983), this type of protein were identified from other lepidopteran insects, such as Corcyra cephalonica (VenkatRao et al., 2016), Cerura vinula (Kayser et al., 2009), Heliothis zea (Haunerland and Bowers, 1986a), Manduca sexta (Riddiford and Hice, 1985) and other lepidopteran insects (Burmester, 2015). Arylphorin-like protein hexamerin was firstly predicted from genomic data of M. sexta, and named according to the
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relatively higher composition of aromatic amine acids (Burmester, 2015). A similar arylphorin is also identified and
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sequenced in S. exigua, blast search in NCBI database revealed that arylphorin-like hexamerins are present in other
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lepidopteran insects, such as Bombyx mori, Plutella xylostella, Pieris rapae, Papilio xuthus, Papilio Machaon, Spodoptera litura, and H. armigera. Averagely, these arylphorin-like hexamerins contain 13.79% aromatic amine acids,
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lower than that of the “true” arylphorins, nevertheless, the methionine content in arylphorin-like hexamerin does
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not change when compared with that of hemocyanins (Figure 3), arylphorin may evolve from arylphorin-like sequence based on the phylogenetic pattern and the characterization of amine acid composition.
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In Diptera there are also the expression of hexamerins rich in aromatic amino acids or methionine (Burmester
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and Scheller, 1995), however, they are not closely related with arylphorins or MRSPs from Lepidoptera according to the phylogenetic tree (Figure 4). Thus, the accumulation of these amino acids in hexamerins in various insect orders
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may occurred independently (Hagner-Holler et al., 2007), which reflect the common requirement of aromatic amino
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acids or methionine for metamorphosis or cuticle formation (Burmester, 2015). It is known that hexamerin is evolved from hemocyanin which act as respiratory protein binding copper, thus oxygen in arthropod. Insect respiration does not depend on hexamerin to transport oxygen, hexamerin lost most of the copper binding sites during the evolution (Burmester, 2002; Burmester, 2004; Pick et al., 2009). In lower insect order, Collembora, six His residues for copper binding remain conserved in hemocyanin, however, hexamerins from Diplura lost most of the His residues in deduced copper binding sites, only two His residues are conserved (site 2 and 5). Arylphorin, MRSP, and MMRSP of lepidopteran insects lost five of the six histidine residues in copper binding sites, 11
ACCEPTED MANUSCRIPT only one His (site 2) is conserved in these types of hexamerin. However, RbH reserve two (site 2 and 3) among six His residues, and some arylphorin-like hexamerins have the same conserved His residues (site 2 and 3) with that of RbH, other arylphorin-like hexamerin only have one reserved His residue at site 3 (Figure S1). The number and sites of reserved His residues in RbH and arylphorin-like hexamerins combined with the phylogenetic tree justify their basal position in hexamerin evolution of Lepidoptera. The evolution and diversity of hexamerin in lepidopteran insects may
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metamorphosis, or distinct functions in this order, which need further study.
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imply the undefined biological backgrounds, reflecting either a special need for protein storage during
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4.2 The structure and function characterizations of hexamerins
In larvae of beet armyworm hexamerins are predominantly expressed in fat body, this expression pattern and
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the structural characteristic with signal peptide suggest they are extracellular proteins and secreted into the
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hemolymph. The hexamerins secreted into hemolymph are primarily considered as storage proteins to provide energy for non-feeding periods, however, recent evidences demonstrate them as versatile molecule (Telfer and
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Kunkel, 1991). The evolution of diverge hexamerins may reflect protein subfunctionalization and the special
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requirement during metamorphosis. Methionine rich hexamerins may support vitellogenesis (Pan and Telfer, 1996; Pan and Telfer, 2001), while hexamerins rich in aromatic amine acids may be preferentially used to build the adult
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cuticle (Lieb et al., 2016; Telfer and Kunkel, 1991). Hexamerins are crucial for insect development, the disruption of
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hamexarin expression has negative effect on the larval development and movement, which may eventually die. Knockdown of SeSP1 (SeMRSP) or SeHex (SeRbH) by dsRNA injection resulted in low adult survival (Tang et al., 2010), suggesting hexamerin expression is crucial for insect life. In termites, hexamerin plays a major role in cast determination in cooperation with juvenile hormone (Zhou et al., 2006). Few studies implicate hexamerins’ involvement in immune response (Coates and Nairn, 2014; Eliautout et al., 2016; Lourenco et al., 2009; Lourenco et al., 2012; Poopathi et al., 2014; Zhu et al., 2009). Hexamerins may bind to small organic metabolites like riboflavin (Magee et al., 1994; Miller and Silhacek, 1995) 12
ACCEPTED MANUSCRIPT with high affinity and also involve into the transportation of hormones such as juvenile hormone (Braun and Wyatt, 1996). The expression of hexamerins were regulated by ecdysone and juvenile hormone (Gkouvitsas and Kourti, 2009; Hathaway et al., 2009; Hwang et al., 2001; Manohar et al., 2010; Spit et al., 2016; VenkatRao et al., 2016; Zhou et al., 2007). The regulation effect may be elicited by binding of hexamerins with juvenile hormone, thus preventing it from eliciting downstream effects on developmental gene expression (Zhou et al., 2006).
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4.3 The effects of insecticide exposure on hexamerin expression
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It was reported the expression of hexamerins were affected by xenobiotics, the larval serum protein 2 was
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upregulated in Culex quinquefasciatus larvae exposed to temephos (Games et al., 2016). Transcriptome analysis demonstrated imidacloprid or permethrin resistant mosquito (Aedes. aegypti) showed higher levels of transcription
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in hexamerin genes (David et al., 2014; Riaz et al., 2013). Hexamerins of the lepidopteran H. zea have been shown to
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bind to lipophilic insecticides, suggesting a putative role in resistance (Haunerland and Bowers, 1986b). However, there were also opposite reports that hexamerins were significantly down-regulated in pine sawyer beetle
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(Monochamus alternatus) when exposed to sublethal chloramine phosphorus (an organophosphorus insecticide) (Lin
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et al., 2013), and arylphorin was down-regulated in Vip3Aa toxin-treated larvae of S. exigua (Bel et al., 2013). The expression levels of two storage protein genes (HexL1 and Lsp1) were down-regulated in spinosad-resistant strain of
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oliver fly (Bactrocera oleae) (Sagri et al., 2014). In present study hexamerin genes are found to be down-regulated in
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larvae of S. exigua exposed to sublethal lambda-cyhalothrin (a pyrethroid insecticide). The different physicochemical properties of xenobiotic or toxins could be the reason for these different observations. The regulation mechanism for insecticide induction of hexamerin expression need further investigation. In summary, experimental identification of five new hexamerin genes in S. exigua increases the gene number of hexamerin in S. exigua to seven. Phylogenetic analysis reveals the presence of arylphorin-like hexamerin and the basal position of RbH in lepidopteran insects. These hexamerins are mainly expressed in fat body and act as the storage protein during metamorphosis, and their expression are influenced by insecticide exposure. These findings 13
ACCEPTED MANUSCRIPT add our understanding on insect hexamerins. Acknowledgement This research was supported by grants from the National Natural Science Foundation of China (Grant number: 31272063). Conflict of Interest statement: The authors declare no conflict of interest.
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SUPPORTING INFORMATION
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Supporting information may be found in the online version of this article. Reference
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ACCEPTED MANUSCRIPT Spodoptera exigua (Lepidoptera: Noctuidae) in Guangdong Province, China. Crop Prot 61, 58-63. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30, 2725-2729. Tang, B., Wang, S., Zhang, F., 2010. Two storage hexamerins from the beet armyworm Spodoptera exigua: cloning, characterization and the effect of gene silencing on survival. BMC Mol Biol 11, 65. Telfer, W.H., Keim, P.S., Law, J.H., 1983. Arylphorin, a new protein from Hyalophora cecropia: Comparisons with calliphorin and manducin. Insect Biochem 13, 601-613. Telfer, W.H., Kunkel, J.G., 1991. The function and evolution of insect storage hexamers. Annu. Rev. Entomol 36, 205-228. Tian, X., Zhao, S., Guo, Z., Hu, B., Wei, Q., Tang, Y., Su, J., 2018. Molecular characterization, expression pattern and metabolic
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Zhu, X., Yuan, M., Shakeel, M., Zhang, Y., Wang, S., Wang, X., Zhan, S., Kang, T., Li, J., 2014. Selection and evaluation of reference genes for expression analysis using qRT-PCR in the beet armyworm Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae).
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ACCEPTED MANUSCRIPT Figure legend Figure 1 The alignment of amino sequences of hexamerins from S. exigua. The sequences were aligned using clusta-W software；The signal peptides predicted using SignalP 4.1 software (http://www.cbs.dtu.dk/services/SingalP) are shaded in black and underlined with double line. The conserved domains were predicted using the CDSearch tool (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Three conserved domains (Hemocyanin N, Hemocyanin M
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and Hemocyanin C) are shaded in yellow, light grey and blue, respectively and are underlined with dot line, broke line
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and solid line, respectively.
Figure 2 Amino acid composition of hexamerin proteins from S. exigua. X-axis represents different amino acids and
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Y-coordinate represents the percentage of different amino acids．
Figure 3 The average content of methionine (A) and aromatic amine acids (B) in five types of hexamerins from
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lepidopteran insects and hemocyanins of Crustaea and Collembola. The sequence information of these hexamerins
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were listed in supplementary Table S3. Different letters on the error bars indicate significant differences among
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hexamerin groups based on ANOVA with Tukey’s HSD multiple comparison test (p < 0.05).
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Figure 4 The maximum likelihood tree of amino acid sequences of hexamerin generated by MEGA6.0 software using Jones-Taylor-Thoraton medel. The phylogenetic tree was calculated on the basis of a multiple sequence alignment of 157 protein sequences. See supplementary Table S3 for a list of abbreviations of these proteins. The hexamerins of S. exigua are marked with red circle.
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ACCEPTED MANUSCRIPT Figure 5 The expression pattern of hexamerin genes in larvae tissues of S. exigua. The expression pattern of hexamerin genes was analyzed in three biological replicates, and each with three technical replicaes, by quantitative RT-PCR. The cycle threshold (Ct) values for hexamerins were normalized to the Ct values of GAPDH, and the relative expression levels of each gene were calculated using 2−ΔΔCT method and normalized to the minimum value for each gene. The results are presented as the mean ± SD (n = 3). Different letters on the error bars indicate significant
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differences among the tissues based on ANOVA with Tukey’s HSD multiple comparison test (p < 0.05).
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Figure 6 The expression changes of hexamerin genes under stress of lambda cyhalothrin in larvae of S. exigua. The expression levels in S. exigua larvae were determined after 24h and 48hexposure at 25 mg/L insecticides. The
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experiments were repeated three times. The results are presented as the mean ± SD (n = 3). * above the error bar
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represents the significant difference between insecticide challenge and the control (p < 0.05).
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ACCEPTED MANUSCRIPT Table 1 The characteristics of hexamerins cloned from S. exigua
Hexamerin
Accession Amide acid Signal peptide
Predicted
number
(aa)
(aa)
Mol mass (kDa)
KY419212
751
16
89.0
SeMMRSPα KY419215
753
18
SeMMRSPβ KY419213
749
SeARYLα
KY419214
SeARYLβ SeRbH
Composition (Mol %) Phe + Tyr
9.07
8.26
9.05
90.3
8.94
4.78
13.15
18
89.5
8.91
4.14
12.69
691
17
82.9
6.25
2.17
19.10
KY419217
691
17
82.8
6.41
2.32
18.53
KY419216
707
19
82.2
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Met
6.33
1.56
10.32
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SeMRSP
PI
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Graphical abstract Highlights: Five new hexamerin genes were identified from Spodopera exigua There are five types of hexamerins based in phylogenetic pattern and amine acid composition RbH is positioned at the base of evolutionary tree of hexamerins in Lepidoptera Hexamerins were down-regulated in larvae exposed to lambda cyhalothrin
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Shiyu Wang, Bo Hu, Qi Wei, Jianya Su PII: DOI: Reference:
S1226-8615(19)30001-9 https://doi.org/10.1016/j.aspen.2019.04.006 ASPEN 1375
To appear in:
Journal of Asia-Pacific Entomology
Received date: Revised date: Accepted date:
1 January 2019 15 March 2019 16 April 2019
Please cite this article as: S. Wang, B. Hu, Q. Wei, et al., Cloning and characterization of hexamerin in Spodoptera exigua and the expression response to insecticide exposure, Journal of Asia-Pacific Entomology, https://doi.org/10.1016/j.aspen.2019.04.006
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ACCEPTED MANUSCRIPT Cloning and characterization of hexamerin in Spodoptera exigua and the expression response to insecticide exposure Shiyu Wang, Bo Hu, Qi Wei, and Jianya Su* Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Nanjing 210095,
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China.
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* Corresponding authors:
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Dr. Jianya Su Department of Pesticide Science,
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College of Plant Protection,
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Nanjing Agricultural University, Nanjing 210095, China.
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Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract Hexamerins are hemolymph-proteins, which are mainly considered as storage proteins for non-feeding stages, and also undertake other roles during insect development and growth, however the characterization of hexamerin proteins in Spodoptera exigua is less understood. In this study five new hexamerin genes were identified and a total seven hexamerin genes were reported in S. exigua. These hexamerins contain the typical domains of hemocyanin at
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the N-terminal, C-terminal and in the middle of their protein sequences. These genes are mainly expressed in fat
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body, and the signal peptide sequences at their N-terminal of protein sequences can drive the expressed protein to
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excrete into hemolymph after synthesis. The phylogenetic analysis and amine acid composition revealed S. exigua express five different types of hexamerins: 1) Storage protein rich in methionine residue (MRSP), 2) Storage protein
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moderately rich in methionine (MMRSP), 3) Hexamerin with high composition of aromatic amino acids (Arylphorin), 4)
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Arylphorin-like hexamerin, and 5) Riboflavin-binding hexamerin (RbH). The phylogenetic pattern combined with the comparison of conserved histidine residues in copper binding sites of hexamerins revealed basal position of RbH and
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the evolutionary pathway in lepidopteran hexamerins. Finally, the induction expression of hexamerins by insecticide,
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lambda-cyhalothrin, were analyzed, results showed that lambda-cyhalothrin exposure may down-regulate their expression. This study increased the gene number of hexamerin to seven, and reported their expression and
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structural characterizations, the finding will facilitate the understand of hexamerin in other insects.
Key words: Hexamerin, Spodoptera exigua, phylogenetic analysis, insecticide induction, lambda-cyhalothrin.
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ACCEPTED MANUSCRIPT 1. Introduction Hexamerins belong to an arthropod protein superfamily that additionally includes hemocyanins, phenoloxidases and hexamerin receptors (Burmester, 2002). There were different nomenclatures for hexamerins according to occurrence in insect species (calliphorin, manduci, etc), their particular amino acid composition (arylphorin, methionine-rich storage protein), or simply referred as “(larval) storage proteins”. The name “hexamerin” refers to
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their composition of six homo- or hetero-hexamer subunits ranging 70 - 90 kDa to form a native molecular mass of
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about 500 kDa (Burmester and Scheller, 1999; Telfer and Kunkel, 1991). Hexamerins resemble the arthropod
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hemocyanins in terms of structure and sequence (Burmester and Schellen, 1996). While hemocyanins serve as respiratory proteins, hexamerins do not bind copper and thus oxygen. Molecular phylogenetic analyses have shown
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that hexamerins evolved within the hexapod lineage, after the divergence from hemocyanin of crustaceans (Pick et
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al., 2009).
Hexamerins have been discovered in all insect species studied so far and are mainly expressed by fat body and
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exported into the hemolymphs (Burmester, 2004). Initially, they have been considered solely as storage proteins that
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provide amino acids and energy for non-feeding periods such as molting process. This view is supported by their massive accumulation during late nymph and larval stages, where they may account for up to 50% of the soluble
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proteins. Hexamerins disappear during metamorphosis (Telfer and Kunkel, 1991) and hexamerin-derived amino acid
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are incorporated into adult tissues (Pan and Telfer, 2001). Analogous to the function of vertebrate serum proteins, hexamerins may transport hormones such as ecdysteroids and juvenile hormone (Telfer and Kunkel, 1991). The regulation of juvenile hormone level by hexamerins may also be instrumental in caste differentiation of termites (Zhou et al., 2007). There is also evidence that at least some hexamerins are involved in immune response to bacteria (Eliautout et al., 2016; Ma et al., 2012; Poopathi et al., 2014), Plasmodium (Lombardo and Christophides, 2016), parasitoid (Zhu et al., 2009), and acclimation (Sonoda et al., 2006). Hexamerins may also bind to other organic compounds such as riboflavin (Magee et al., 1994; Miller and Silhacek, 1995; Pan and Telfer, 1999) or certain 3
ACCEPTED MANUSCRIPT insecticides (Haunerland and Bowers, 1986b). Hexamerin was found to be inducible by Cry1Ac toxin in Heliceverpa armigera, and the binding of hexamerin with toxin leads to the formation of insoluble aggregates, which may hamper the toxicity of Bt toxins (Ma et al., 2005). An 80-kDa protein, which was idenfied as hexamerin, was found to be highly expressed in Culex quinquefasciatus resistant to toxin of Bacillus sphaericus, and. binding of toxins with this protein possibly lead to the toxin sequestration, however, the role of hexamerin expression in toxin resistance
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remains elusive (Poopathi et al., 2014).
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Spodoptera exigua is an important insect pest on vegetable crops, the frequent applications of insecticides have
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resulted in rapid development of insecticide resistance in the field populations of this pest from worldwide (Su and Sun, 2014). In present research the genes for hexamerins in S. exigua were cloned and analyzed for the expression
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level in different tissues of larvae. We obtained seven hexamerin cDNA sequences which encode proteins with typical
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of hexamerin domains. The expression changes of six hexamerin genes in larvae under insecticide challenge were analyzed and the function and phylogenetic of hexamerins was also discussed.
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2. Materials and methods
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2.1 Insects
A laboratory strain of S. exigua, which was obtained from Wuhan Kernel Biopesticide Company, Wuhan, China,
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in May 2001, was used in this study. This strain was maintained in laboratory without exposure to any insecticide. All
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stages of insects were maintained in an insectary at 27 ± 1°C under a photoperiod of 16:8 (L:D) and 50 % - 70 % RH. The larvae were reared with artificial diet and adults were fed 10 % honey solution (Lai and Su, 2011) 2.2 RNA extractions and cDNA synthesis For gene cloning the third instar larvae of S. exigua were collected, and total RNAs were extracted using TRIZOL regent (Invitrogen. USA) following the manufacturer’s instructions. The quality of total RNA was check by agarose gel electrophoresis and the quantity of total RNA was determined with NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). First-strand cDNA was synthesized using Supermo III RT Kit (Bio Teke, China). 4
ACCEPTED MANUSCRIPT 2.3 Cloning and sequence analysis of hexamerins According to the transcriptome data of S. exigua (Li et al., 2013; Pascual et al., 2012) the sequences of seven hexamerin genes were verified by gene clone and sequencing. The gene-specific primers were designed to directly amplify the nucleotide sequence for each gene’s ORF (Table S1). PCRs were made using Taq Master Mix (Vazyme Biotech Co., Ltd. Nanjing, China) according to following PCR protocol: 1 cycle of denaturation at 95 °C for 3 min, 35
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amplification cycles (94 °C, 50 s; 50 °C, 50 s; 72 °C, 1 min), and finally extention at 72 °C for 10 min. The PCR
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reactions were made in 50 μL system containing PCR reagents according to the instructions. PCR products were
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electrophoresed on 1.5 % agarose gel, extracted by gel extraction kit (E.Z.N.ATM Gel Extration Kit) and were subcloned into pMD19-T vector (Takara, Dalian, China). The positive clones were sent for sequencing by GENEWIZ
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Biological technology CO., Ltd (Suzhou, China).
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Sequence alignment was made using Clustal X6.0 software. Signal peptides were predicted using the web service of http://www.cbs.dtu.dk/services/SignalP/, and the CD search tool of NCBI was used to predict the structural
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domains of proteins. The maximum likelihood phylogenetic tree (ML tree) of hexamerins was constructed using
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amino acid sequences of hexamerins from S. exigua and other insect orders by MEGA6.0. Bootstrap analysis was performed using 1000 replicates to evaluate the significance of the nodes (Tamura et al., 2013).
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2.4 Quantitative real-time PCR (qRT-PCR)
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For analysis pattern of hexamerin genes, hemocytes of 1-day-old 5th instar larvae were collected and their head, fat body, midgut, and epidermis were dissected according to previous protocols (Hu et al., 2019; Kim and Kim, 2010; Tian et al., 2018). Total RNAs were isolated using TRIZOL regent and subjected to DNase I to remove any residual genomic DNA according to the manufacturer’s instructions. The isolated RNAs were reverse transcribed using the HiScriptTM Q Select RT SuperMix (Vazyme, Nanjing, China). The mRNA abundance of hexamerin genes was estimated by qRT-PCR in an ABI 7300 Fast Real-Time PCR System (Applied Biosytems, Foster City, CA) using AceQ qPCR SYBR Green Master Mix kit (Vazyme, China) according to the instruction. GAPDH was used as internal reference gene for 5
ACCEPTED MANUSCRIPT analyses (Zhu et al., 2014). The PCR primers for hexamerins were designed using the Primer 5 software. The specificity and sensitivity of primers for qRT-PCR assays were evaluated through melting curve analysis coupled with agarose gel electrophoresis (distinct single peaks in melting curve analysis, unique clear PCR product in agarose gel electrophoresis). PCR efficiencies were calculated from the standard curve (Table S2). A reverse transcription negative control (without reverse transcriptase) and a non-template negative control were included for each primer
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set to confirm the absence of genomic DNA and to check for primer-dimer or contamination in the reactions,
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respectively. The PCR system contained 0.8 µL of the forward and reverse primers (10 mmol/L), 10 µL 2 x SYBR Green
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Master Mix, 0.4 µL ROX X Reference Dye2, 1 µL of the diluted cDNA samples and nuclease-free water in a final volume of 20 µL. The quantification analysis for each gene was made in three biological samples (n = 3) and every
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biological sample was assayed with three mechanical repeats. The reaction conditions were as follows: an initial
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denaturation step of 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60 °C for 40 s. Melt curve analysis of the products was as follows: heating to 95°C for 15 s, decrease to 60°C for 60s and then 95°C for 15 s. Relative
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expression levels of these genes were calculated by the 2-∆∆T method (Livak and Schmittgen, 2001). All method and
guidelines (Bustin et al., 2009).
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2.5 Insecticide exposure
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data were confirmed to follow the minimum information for publication of quantitative real time PCR experiments
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In the research of gene expression responses in insects exposed to insecticides a sublethal concentration of insecticide was applied to challenge insects to avoid the high mortality at high concentration of insecticide. For insecticide induction studies in this study the 3rd instar larvae of S. exigua were exposed to LC20 concentration (0.25mg/L) of lambda-cyhalothrin (Jiangsu Yangnong Chemical Company, China) and collected after insecticide exposure for 24 h and 48 h. The total RNAs were isolated as previous protocol. Forty larvae were used as one treatment, and three replicates were applied in insecticide exposure experiment, therefore, totally 120 larvae were exposed to insecticide, and then among the alive larvae half were harvested at 24 h, and the remaining alive larvae 6
ACCEPTED MANUSCRIPT at 48 h for RNA isolation. The total RNA isolated from ten individual larvae were pooled as one biological sample for quantitative real-time PCR according to the procedure described in 2.4 section.
3. Results 3.1 The identification and characterization of hexamerins from S. exigua Seven hexamerin genes were identified from transcriptome sequences and further confirmed through RT-PCR
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and sequencing from larvae of S. exigua, the structure information including theoretical isoelectric point and the
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theoretical molecular weight of proteins coding by six sequences with complete open read frame (ORF) are listed in
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Table 1. The seventh one is a partial sequence lacking ORF, therefore its structure information is not listed. The six hexamerin genes encode proteins with 691- 753 amino acids; each protein contains signal peptide of 16 - 19 amino
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acids at N-terminal. Three conserved hemocyanin domains are found at the N-terminal, C-terminal of sequences and
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in the middle of sequences, respectively (Figure 1), suggesting they belong to hemocyanin family protein. Different from hemocyanins, hexamerin proteins lost most of the copper binding sites in sequences, we compare the copper
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binding sites of hexamerins from S. exigua with that from other lepidoptera insects, springtail, dipluran and crustean
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(Figure S1). The hemocyanins of lobsters in Crustean and springtails in Collembola keep these six conserved histidine (His) residues for copper binding sites, Dipluran lost four of the six, SeRbH of S. exigua also lost four of the six His
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residues, however, only one His residue are conserved in other six hexamerins from S. exigua (Figure S1).
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Hexamerins from S. exigua have different composition of amino acids, protein encoded by KY419212 contains relatively high content of methionine (8.26 %), therefore belongs to methionine-rich soluble protein group (MRSP). Proteins encoded by KY419215 and KY419213 contains moderate content of methionine, 4.78 % and 4.14 % respectively, hence belongs to the moderately methionine-rich soluble protein group (MMRSP). KY419214 and KY419217 encode protein with high composition of aromatic amino acids (phenylanine and tyrosine), 19.10 % and 18.53 %, respectively (arylphorin, ARYL). KY419216 encodes protein (RbH) with very low composition of methionine (1.56 %) (Figure 2). The average content of methionine and aromatic amine acids were also analyzed for the five 7
ACCEPTED MANUSCRIPT types of hexamerins from lepidopteran insects and compared with that of hemocyanin from Crustaea and Collembora (Figure 3). The average percentage of methionine is 1.70 %, 2.4 % and 2.25 % in RbH, ARYL and ARYL-like hexamerins, respectively, which is similar with that of lobsters (2.28 %) and springtails (2.21 %). However, there is a significant increase of methionine content in MMRSP (4.92 %), and a further obvious increase in MRSP (7.94 %) (Figure 3A). The mean content of aromatic amine acids in RbH is 10.62 %, it is also similar with that of lobsters
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(9.78 %) and springtails (8.76 %). Their content is increased significantly in ARYL-like hexamerin (13.79 %), and a
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further significant increase is observed in ARYL (18.39 %). However, it is decreased in MMRSP significantly (11.5 %),
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and then slightly lowered down to 9.62 % in MRSP (Figure 3B). 3.2 The phylogenetic analysis of hexamerins
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To understand the evolution of hexamerins we collected the hexamerin sequences of insects available from the
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NCBI database. Phylogenetic tree reconstructions were carried out using maximum likelihood method based on the amino acid sequences. No hexamerin is found in the available sequences of Crustacea, and hexamerins appear in the
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order of Collembora, which is positioned at the base of hexamerin tree, and hemocyanin and hexamerin coexist in
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this order. The hexamerins from Hemiptera are close to that of Apteygota. Hexamerins from holometabolous insects form single clades in each order except Lepidoptera. In lepidopteran insects hexamerins are split into two diverge
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that of Diptera (Figure 4).
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clades, one small branch (RbH) and big branch with other four types of hexamerins. The big branch is parallel with
Based on the ML tree and amino acid composition the hexamerins from lepidoptera insects are divided into five groups: MRSP protein belongs to the methionine-rich hexamerins group; MMRSPα and MMRSPβ belong to the moderately methionine-rich hexamerins group; ARYLα and ARYLβ belong to the arylphorins group; RbH belong to the riboflavin-binding hexamerins group and an ARYL-like group. RbH is positioned at the root of lepidopteran hexamerin clade, and then the ARYL-like hexamerin, which was first predicted from Manduca sexta by Burmester in 2015. This sequence was also identified in S. exigua. And further evolution give rise to ARYL and MMRSP/MRSP hexamerins 8
ACCEPTED MANUSCRIPT (Figure 4). 3.3 Expression pattern of hexamerins in S. exigua larvae In the first stage of the study we find only six hexamerin gene in S. exigua, the arylphorine-like hexamerin was searched out when we blasted insect hexamerins in the NCBI database to construct the phylogenetic tree, therefore the expression profiles of only six hexamerin genes were determined in the larvae of beet armyworm by qRT-PCR.
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The relative expression level of mRNAs was measured for each of them in the head, fat body, midgut, epidermis and
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hemocytes of the 1-day-old 5th instar larvae. The results show that the expressions are detected in all the analyzed tissues for six hexamerins, however, each hexamerin has specific expression pattern in different tissues of larvae
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(Figure 5). SeMRSP has higher expression level in fat body and epidermis than in other tissues. For SeMMRSP the
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highest expression level is observed in fat body, and then in hemocytes, and low level of expression is observed in
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head, epidermis and midgut. For SeMMRSP the highest expression is found in fat body and hemocytes, and very low expression in head, epidermis and midgut. The rank of expression levels for SeAYRL is that: fat body > head >
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epidermis, hemocyte > midgut. The ranking for SeAYRL is fat body > epidermis > hemocyte, head, and midgut. Fat
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body and hemocytes have higher expression of SeRbH than other tissues, and midgut has the lowest expression of this gene. The common characteristic for expression is that six hexamerins are highly expressed in fat body, however
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exigua.
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very low in midgut of larvae. This result demonstrates that fat body is the main expression tissue for hexamerin in S.
3.4 Expression response of hexamerins to insecticide challenges To understand the responses of hexamerin expression in S. exigua challenged by insecticide qRT-PCR were performed to determine the changes in transcriptional level for each of the hexamerin genes. Lambda-cyhalohrin exposure at the LC20 concentration (0.25 mg/L) down-regulates the expression of these hexamerins. The expression levels of SeMRSP, SeMMRSP and SeARYL are significantly reduced at 24h and 48h exposure post. SeARYL is down-regulated at 48h exposure post. The expressions of SeRbH and SeMMRSP are slightly reduced at 24h, and then 9
ACCEPTED MANUSCRIPT recover to the control level at 48h (Figure 6). The expressions of hexamerin genes in larvae of S. exigua are influenced by the insecticide challenge.
4. Discussion 4.1 The phylogenetic analysis of hexamerins within insects In a previous report two hexamerin genes (SeHex and SeSP1) were cloned and characterized from S. exigua
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(Tang et al., 2010), in this study we identified other five hexamerins, which were not reported previously in this
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species, totally seven hexamerin genes of S. exigua were identified. Before our study only six hexamerin genes had
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been identified in Manduca sexta, and were classified into five distinct groups (Burmester, 2015). The hexamerins from S. exigua are also belonging to these five groups based on amino acid composition and evolutionary analysis
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(Figure 4).
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Hexamerins have recently been applied in the phylogenetic analysis of insect orders, and the trees constructed from insect hexamerins are consistent with the generally accepted relationships of insect taxa, however, these
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analysises were mainly made on hexamerins from “lower” insects (Burmester, 2002; Burmester, 2004; Burmester et
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al., 1998; Hagner-Holler et al., 2007; Pick et al., 2009; Xie and Luan, 2014; Zhang et al., 2017). Here we incorporated seven hexamerin sequences of S. exigua into the phylogenetic analysis and exhibited an evolutionary pattern
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different from previous one (Burmester, 2015; Burmester et al., 1998). The ML tree demonstrates the complex
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evolution of hexamerins in Lepidoptera, which exceeds the diversity of other insect orders. The diversity may be partly due to an insufficient understanding on hexamerins in other insect taxa, or the biased sequencing approaches (Burmester et al., 1998).
RbH is positioned at the base of lepidopteran clade of hexamerins, and the content of methionine or aromatic amine acids in RbH is much similar with that of hemocyanin from Crustaea and Collembora, mightly suggesting RbH is the archetype of hexamerin in Lepidoptera. Different from other insect orders, lepidopteran evolution gave rise to the distinct aromatic or methionine-rich hexamerins to cope with undefined requirement during metamorphosis in 10
ACCEPTED MANUSCRIPT this order. Arylphorin was firstly identified in Hyalophora cecropia (Lepidoptera) and named due to the high content of aromatic amino acids (Telfer et al., 1983), this type of protein were identified from other lepidopteran insects, such as Corcyra cephalonica (VenkatRao et al., 2016), Cerura vinula (Kayser et al., 2009), Heliothis zea (Haunerland and Bowers, 1986a), Manduca sexta (Riddiford and Hice, 1985) and other lepidopteran insects (Burmester, 2015). Arylphorin-like protein hexamerin was firstly predicted from genomic data of M. sexta, and named according to the
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relatively higher composition of aromatic amine acids (Burmester, 2015). A similar arylphorin is also identified and
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sequenced in S. exigua, blast search in NCBI database revealed that arylphorin-like hexamerins are present in other
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lepidopteran insects, such as Bombyx mori, Plutella xylostella, Pieris rapae, Papilio xuthus, Papilio Machaon, Spodoptera litura, and H. armigera. Averagely, these arylphorin-like hexamerins contain 13.79% aromatic amine acids,
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lower than that of the “true” arylphorins, nevertheless, the methionine content in arylphorin-like hexamerin does
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not change when compared with that of hemocyanins (Figure 3), arylphorin may evolve from arylphorin-like sequence based on the phylogenetic pattern and the characterization of amine acid composition.
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In Diptera there are also the expression of hexamerins rich in aromatic amino acids or methionine (Burmester
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and Scheller, 1995), however, they are not closely related with arylphorins or MRSPs from Lepidoptera according to the phylogenetic tree (Figure 4). Thus, the accumulation of these amino acids in hexamerins in various insect orders
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may occurred independently (Hagner-Holler et al., 2007), which reflect the common requirement of aromatic amino
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acids or methionine for metamorphosis or cuticle formation (Burmester, 2015). It is known that hexamerin is evolved from hemocyanin which act as respiratory protein binding copper, thus oxygen in arthropod. Insect respiration does not depend on hexamerin to transport oxygen, hexamerin lost most of the copper binding sites during the evolution (Burmester, 2002; Burmester, 2004; Pick et al., 2009). In lower insect order, Collembora, six His residues for copper binding remain conserved in hemocyanin, however, hexamerins from Diplura lost most of the His residues in deduced copper binding sites, only two His residues are conserved (site 2 and 5). Arylphorin, MRSP, and MMRSP of lepidopteran insects lost five of the six histidine residues in copper binding sites, 11
ACCEPTED MANUSCRIPT only one His (site 2) is conserved in these types of hexamerin. However, RbH reserve two (site 2 and 3) among six His residues, and some arylphorin-like hexamerins have the same conserved His residues (site 2 and 3) with that of RbH, other arylphorin-like hexamerin only have one reserved His residue at site 3 (Figure S1). The number and sites of reserved His residues in RbH and arylphorin-like hexamerins combined with the phylogenetic tree justify their basal position in hexamerin evolution of Lepidoptera. The evolution and diversity of hexamerin in lepidopteran insects may
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metamorphosis, or distinct functions in this order, which need further study.
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imply the undefined biological backgrounds, reflecting either a special need for protein storage during
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4.2 The structure and function characterizations of hexamerins
In larvae of beet armyworm hexamerins are predominantly expressed in fat body, this expression pattern and
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the structural characteristic with signal peptide suggest they are extracellular proteins and secreted into the
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hemolymph. The hexamerins secreted into hemolymph are primarily considered as storage proteins to provide energy for non-feeding periods, however, recent evidences demonstrate them as versatile molecule (Telfer and
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Kunkel, 1991). The evolution of diverge hexamerins may reflect protein subfunctionalization and the special
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requirement during metamorphosis. Methionine rich hexamerins may support vitellogenesis (Pan and Telfer, 1996; Pan and Telfer, 2001), while hexamerins rich in aromatic amine acids may be preferentially used to build the adult
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cuticle (Lieb et al., 2016; Telfer and Kunkel, 1991). Hexamerins are crucial for insect development, the disruption of
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hamexarin expression has negative effect on the larval development and movement, which may eventually die. Knockdown of SeSP1 (SeMRSP) or SeHex (SeRbH) by dsRNA injection resulted in low adult survival (Tang et al., 2010), suggesting hexamerin expression is crucial for insect life. In termites, hexamerin plays a major role in cast determination in cooperation with juvenile hormone (Zhou et al., 2006). Few studies implicate hexamerins’ involvement in immune response (Coates and Nairn, 2014; Eliautout et al., 2016; Lourenco et al., 2009; Lourenco et al., 2012; Poopathi et al., 2014; Zhu et al., 2009). Hexamerins may bind to small organic metabolites like riboflavin (Magee et al., 1994; Miller and Silhacek, 1995) 12
ACCEPTED MANUSCRIPT with high affinity and also involve into the transportation of hormones such as juvenile hormone (Braun and Wyatt, 1996). The expression of hexamerins were regulated by ecdysone and juvenile hormone (Gkouvitsas and Kourti, 2009; Hathaway et al., 2009; Hwang et al., 2001; Manohar et al., 2010; Spit et al., 2016; VenkatRao et al., 2016; Zhou et al., 2007). The regulation effect may be elicited by binding of hexamerins with juvenile hormone, thus preventing it from eliciting downstream effects on developmental gene expression (Zhou et al., 2006).
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4.3 The effects of insecticide exposure on hexamerin expression
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It was reported the expression of hexamerins were affected by xenobiotics, the larval serum protein 2 was
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upregulated in Culex quinquefasciatus larvae exposed to temephos (Games et al., 2016). Transcriptome analysis demonstrated imidacloprid or permethrin resistant mosquito (Aedes. aegypti) showed higher levels of transcription
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in hexamerin genes (David et al., 2014; Riaz et al., 2013). Hexamerins of the lepidopteran H. zea have been shown to
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bind to lipophilic insecticides, suggesting a putative role in resistance (Haunerland and Bowers, 1986b). However, there were also opposite reports that hexamerins were significantly down-regulated in pine sawyer beetle
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(Monochamus alternatus) when exposed to sublethal chloramine phosphorus (an organophosphorus insecticide) (Lin
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et al., 2013), and arylphorin was down-regulated in Vip3Aa toxin-treated larvae of S. exigua (Bel et al., 2013). The expression levels of two storage protein genes (HexL1 and Lsp1) were down-regulated in spinosad-resistant strain of
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oliver fly (Bactrocera oleae) (Sagri et al., 2014). In present study hexamerin genes are found to be down-regulated in
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larvae of S. exigua exposed to sublethal lambda-cyhalothrin (a pyrethroid insecticide). The different physicochemical properties of xenobiotic or toxins could be the reason for these different observations. The regulation mechanism for insecticide induction of hexamerin expression need further investigation. In summary, experimental identification of five new hexamerin genes in S. exigua increases the gene number of hexamerin in S. exigua to seven. Phylogenetic analysis reveals the presence of arylphorin-like hexamerin and the basal position of RbH in lepidopteran insects. These hexamerins are mainly expressed in fat body and act as the storage protein during metamorphosis, and their expression are influenced by insecticide exposure. These findings 13
ACCEPTED MANUSCRIPT add our understanding on insect hexamerins. Acknowledgement This research was supported by grants from the National Natural Science Foundation of China (Grant number: 31272063). Conflict of Interest statement: The authors declare no conflict of interest.
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ACCEPTED MANUSCRIPT Figure legend Figure 1 The alignment of amino sequences of hexamerins from S. exigua. The sequences were aligned using clusta-W software；The signal peptides predicted using SignalP 4.1 software (http://www.cbs.dtu.dk/services/SingalP) are shaded in black and underlined with double line. The conserved domains were predicted using the CDSearch tool (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Three conserved domains (Hemocyanin N, Hemocyanin M
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and Hemocyanin C) are shaded in yellow, light grey and blue, respectively and are underlined with dot line, broke line
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and solid line, respectively.
Figure 2 Amino acid composition of hexamerin proteins from S. exigua. X-axis represents different amino acids and
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Y-coordinate represents the percentage of different amino acids．
Figure 3 The average content of methionine (A) and aromatic amine acids (B) in five types of hexamerins from
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lepidopteran insects and hemocyanins of Crustaea and Collembola. The sequence information of these hexamerins
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were listed in supplementary Table S3. Different letters on the error bars indicate significant differences among
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hexamerin groups based on ANOVA with Tukey’s HSD multiple comparison test (p < 0.05).
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Figure 4 The maximum likelihood tree of amino acid sequences of hexamerin generated by MEGA6.0 software using Jones-Taylor-Thoraton medel. The phylogenetic tree was calculated on the basis of a multiple sequence alignment of 157 protein sequences. See supplementary Table S3 for a list of abbreviations of these proteins. The hexamerins of S. exigua are marked with red circle.
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ACCEPTED MANUSCRIPT Figure 5 The expression pattern of hexamerin genes in larvae tissues of S. exigua. The expression pattern of hexamerin genes was analyzed in three biological replicates, and each with three technical replicaes, by quantitative RT-PCR. The cycle threshold (Ct) values for hexamerins were normalized to the Ct values of GAPDH, and the relative expression levels of each gene were calculated using 2−ΔΔCT method and normalized to the minimum value for each gene. The results are presented as the mean ± SD (n = 3). Different letters on the error bars indicate significant
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differences among the tissues based on ANOVA with Tukey’s HSD multiple comparison test (p < 0.05).
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Figure 6 The expression changes of hexamerin genes under stress of lambda cyhalothrin in larvae of S. exigua. The expression levels in S. exigua larvae were determined after 24h and 48hexposure at 25 mg/L insecticides. The
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experiments were repeated three times. The results are presented as the mean ± SD (n = 3). * above the error bar
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represents the significant difference between insecticide challenge and the control (p < 0.05).
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ACCEPTED MANUSCRIPT Table 1 The characteristics of hexamerins cloned from S. exigua
Hexamerin
Accession Amide acid Signal peptide
Predicted
number
(aa)
(aa)
Mol mass (kDa)
KY419212
751
16
89.0
SeMMRSPα KY419215
753
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SeMMRSPβ KY419213
749
SeARYLα
KY419214
SeARYLβ SeRbH
Composition (Mol %) Phe + Tyr
9.07
8.26
9.05
90.3
8.94
4.78
13.15
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89.5
8.91
4.14
12.69
691
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82.9
6.25
2.17
19.10
KY419217
691
17
82.8
6.41
2.32
18.53
KY419216
707
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82.2
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Met
6.33
1.56
10.32
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SeMRSP
PI
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Graphical abstract Highlights: Five new hexamerin genes were identified from Spodopera exigua There are five types of hexamerins based in phylogenetic pattern and amine acid composition RbH is positioned at the base of evolutionary tree of hexamerins in Lepidoptera Hexamerins were down-regulated in larvae exposed to lambda cyhalothrin
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