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Entomological Research 43 (2013) 344–352

RESEARCH PA P E R

Partial sequence and characterization of nitric oxide synthase gene from Hyphantria cunea Hong Ja KIM1,*, Yong Il KIM2,*, Yong Min KWON3, Young Jin KANG4, Hyang Mi CHEON5, Nam Gyu HA6,7 and Sook Jae SEO1 1 Department of Biology, Gyeongsang National University, Jinju, Korea 2 Department of Stem Cells, SMART Institute of Advanced Biomedical Science, Konkuk University, Seoul, Korea 3 Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology, Ansan, Korea 4 Department of Pharmacology, Yeungnam University, Daegu, Korea 5 Division of Research Planning and Coordination, Korea Forest Research Institute, Seoul, Korea 6 Agricultural Experiment and Research Bureau, Gyeongnam, Korea 7 Agricultural Research and Extension Services, Jinju, Korea

Correspondence Sook Jae Seo, Department of Biology, College of Natural Sciences, Gyeongsang National University, Jinju, 660-701, Korea. Email: [email protected] Received 22 April 2013; accepted 26 September 2013. *Contributed equally to this work. doi: 10.1111/1748-5967.12040

Abstract Nitric oxide synthase (NOS) gene has been partially sequenced from Hyphantria cunea and compared with those already determined from insects. Hyphantria cunea NOS possesses putative recognition sites for co-factors heme, BH4, CaM, FMN, FAD, and NADPH common to NOS. The deduced amino acid sequence of H. cunea NOS cDNA showed 70.3% identity to Manduca sexta NOS and 57.6– 69.5% identity to NOS sequences from other insects. Nitric oxide synthase is expressed in all tissues of H. cunea, except in hemocytes. The NOS expression in midgut, fat body, epidermis, and Malpighian tubule strongly increased against Gram-positive and Gram-negative bacterial infection. These results suggest that NOS may play an important role in insect defense system against bacterial infection. Key words: Hyphantria cunea, nitric oxide, Nitric oxide synthase.

Introduction Nitric oxide (NO) is a second messenger molecule in mammals with diverse physiological and pathophysiological roles which include the regulation of vascular tone, neuronal transmission, and anti-tumoral and anti-microbial activities (Moncada et al. 1991). Nitric oxide is a highly reactive free radical gas produced by the conversion of L-arginine into L-citrulline by nitric oxide synthase (NOS) (Knowles & Moncada 1994). Nitric oxide synthase is known to require co-factors such as NADPH, FAD, BH4, calmodulin and Ca2+ to express its activity. Three NOS isoforms, namely neural NOS, inducible NOS and endothelial NOS, have been identified in mammals, including human (Nathan & Xie 1994).

Nitric oxide has been studied in several insects and is known to be involved in many physiological reactions (Davies 2000). It regulates retinal patterning in the optic lobe of Drosophila (Gibbs & Truman 1998) and mediates communication between olfactory receptor, neurons and glomeruli of the antennal lobe in Manduca sexta (Nighorn et al. 1998). Nitric oxide synthase is involved in the function of olfactory receptor cells in fleshfly (Wasserman & Itagaki 2003) and plays an important role in distinct long-term memory of Apis mellifera (Müller 1996). Nitric oxide may be the signal for control of juvenile hormone release from the corpora allata of grasshoppers (Wirmer & Heinrich 2011). In Baculum extradentatum, NO independently regulates heart rate devoid from any nervous system input (Da Silva et al.

© 2013 The Authors Entomological Research © 2013 The Entomological Society of Korea and Wiley Publishing Asia Pty Ltd

Characterization of NOS gene from Hc

2012). Additionally, NO regulates cell proliferation during tissue and organ morphogenesis in Drosophila (Kuzin et al. 1996; Enikolopov et al. 1999). The role of NO and NOS in limiting the impact of pathogens has been studied in insects. For example, Anopheles mosquitoes limit plasmodium parasite infection via inducible synthesis of NO (Dimopoulos et al. 1998; Luckhart et al. 1998; Lim et al. 2005). A similar role of NOS in immunity has been reported in other insects. The proventriculus from immune challenged flies has higher NOS and NO activities (Hao et al. 2003). Nitric oxide is involved in the host immune response by recruiting hemocytes to sites of infection in Drosophila (Carton et al. 2009) and NOS activity is stimulated in the immunecompetent lepidopteran hemocyte line (Weiske & Wiesner 1999). In Drosophila, NO mediates an early stage of the signal transduction pathway, inducing the innate immune response upon gram-negative bacterial infection (Foley & O’Farrell 2003). Nitric oxide synthase cDNAs have been cloned from several insects including D. melanogaster (Regulski & Tully 1995), Rhodnius prolixus (Yuda et al. 1996), Anopheles stephensi (Luckhart et al. 1998), M. sexta (Nighorn et al. 1998), Luciola lateralis (Ohtsuki et al. 2008) and Daphnia magna (Labbé et al. 2009). The cofactorbinding domains are particularly well conserved in these NOSs. In order to understand Hyphantria cunea immune responses against pathogens, we made an attempt to study the Hyphantria NOS gene. The fall webworm, H. cunea, is a polyphagus pest that feeds on about 160 species of broad leaf trees. It tend to damage roadside and garden trees around urban areas rather than in the mountain forests of Korea (Lee & Chung 1998), and can serve as a model of pest control research for polyphagus insects. The present study describes cDNA cloning and characterization of the NOS gene from, H. cunea. This study also focuses on characteristics of NOS genes from H. cunea, their differential accumulation and expression profiles in various tissues upon bacterial infection.

Materials and methods Experimental animals Fall webworms, H. cunea, were obtained from a colony in the Laboratory of Insect Conservation, Department of Sericulture and Entomology, National Institute of Agricultural Science and Technology, Suwon, Republic of Korea. They were reared on an artificial diet (Ito & Tanaka 1960) at 27°C and 75% relative humidity with a photoperiod of 16 h light : 8 h dark (LD 16:8). Under the laboratory conditions, the pupal stage lasts 10 days.

Bacteria Escherichia coli K112 and Enterococcus faecalis KCTC5029 were obtained from Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea. Bacterial infection Bacteria were grown in tryptic soy broth (Difco Lab., Livonia, MI, USA). For immunization, bacterial cells were spun down from 50 mL overnight cultures washed with insect saline buffer (ISB) (128 mM NaCl, 1.8 mM CaCl2, 1.3 mM KCl, 2.3 mM NaHCO3, [pH 6.2]), and resuspended in ISB. Twenty five four-day-old last instar larvae were injected with 1 × 104 viable log-phase bacteria at 5 μL per individual between the first and second abdominal segments using a sterile needle. As a wounding control, a separate set of larvae were injected with 5 μL ISB. After injection, all larvae were covered directly with paraffin and were incubated at 27°C for 1 h, 3 h, 6 h, 12 h and 24 h. Bacterial infection were performed in triplicate in standard cages. At the end of the incubation period, all samples were immediately frozen in liquid nitrogen and stored at −80°C until protein extraction. Protein was extracted from whole body (20 larvae) and Western blot analysis was subsequently performed using the extracted protein samples. Isolation of RNA Total RNA was isolated from fat body tissue of four-day-old last instar larvae using the RNeasy mini kit (Qiagen Inc. Chatsworth, CA, USA) according to manufacturer instructions. All RNA samples were evaluated in agarose gels to ensure that they contained intact rRNA and were free of contaminating DNA. Degenerate primer design and RT-PCR Partial cDNA coding for NOS in H. cunea was obtained by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) PCR. Total RNA was isolated from last instar whole larvae and 1 μg of RNA preparation was reverse transcribed by the Superscript II reverse transcriptase (Gibco BRL, Grand Island, NY, USA) using oligo (dT) 12–18 primers in total reaction volume of 25 μL. The reaction mixture was incubated at 70°C for 5 min, 42°C for 1 h, and at 94°C for 1 min to synthesize cDNA. The obtained cDNA was used in as template for PCR. Degenerate primers were designed based on the highly conserved amino acid residues of NOS of various insects using DNASTAR software. We found highly conserved amino acid residue of NOS of various insects using the DNASTAR program (DNASTAR Inc., Madison,

Entomological Research 43 (2013) 344–352 © 2013 The Authors. Entomological Research © 2013 The Entomological Society of Korea and Wiley Publishing Asia Pty Ltd

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WI, USA). The degenerate primer sequences used in the present study were 5′-CAYTTYAARCARATHGC-3′ (NOS, forward), and 5′- AYGGNCCNGAYTAC-3′ (NOS, reverse). PCR was performed for 35 cycles using cycling conditions of 94°C for 30 s, 53°C for 1 min, and 72°C for 1 min. The resulting 960 bp PCR product was separated on a 1% agarose gel. This fragment was excised from the agarose gel, purified and ligated into a pGEM T-easy vector (Promega, Madison, WI, USA), followed by transformation into JM109 competent cells (Promega). Several clones were isolated and then the nucleotide sequence was determined using a BigDye Terminator Cycle Sequencing Kit in an automated DNA sequencer (ABI 3730XL; Applied Biosystems, Foster City, CA, USA). Rapid amplification of cDNA end PCR To obtain the 5′-end of H. cunea NOS open reading frame, 5′- rapid amplification of cDNA end (5′-RACE) PCR was performed as per manufacturer’s instructions (Gibco-BRL, Grand Island, NY, USA) Gene-specific primer and nested primer (A and B) were synthesized as follows: A, 5′- TACGATCATAAGGGAG -3′ and B, 5′ACTAAGCTCCAGTACAC -3′. Total RNA was extracted from whole body using RNeasy mini kit (Qiagen, Inc., Chatsworth, CA, USA (complete address) according to the manufacturer’s instruction. From 5 μg of total RNA, single-stranded cDNA was synthesized using superscript reverse transcriptase (Gibco-BRL) and gene-specific primer A. After cDNA synthesis, the product was purified using the Glass MAX Spin Cartridge, and was tailed with dCTP and TdT. The tailed cDNA was amplified with the abridged anchor primer and nested gene-specific primer B, and then re-amplified using the same primers. For 3′-RACE, 3 μg of total RNA was reverse transcribed according to the protocol recommended by the manufacturer (Bioneer, Daejeon, Korea). The resulting cDNA was amplified with an adaptor primer and a gene-specific primer, 5′TCTACGTATGCGGAGAC -3′. Re-amplified product was separated by agarose gel electrophoresis. The resulting 5′/3′ RACE PCR products were cloned into the pGEM T-easy vector (Promega) and sequenced. Analysis of sequence data The complete coding sequence of H. cunea NOS was compared with the GenBank database using the program BLASTX. The NOS amino acid sequences were sourced from GenBank and were used for sequence alignment and phylogenetic analysis. Editing and analysis of DNA sequence data was performed with DNASTAR software using the MEGALIGN program to generate pairwise alignment. 346

Preparation of antiserum Polyclonal antibodies produced in rabbit against iNOS (DB BIOTECH, Kosice, Slovakia) and actin (Sigma-Aldrich, St. Louis, USA) were purchased. SDS-PAGE and Western blot SDS-PAGE (12.5%) was performed according to the method of Laemmli (1970). Samples at a concentration of 20 μg were heated at 90°C for 9 min in the presence of 2% SDS and 5% β-mercaptoethanol. Following SDS-PAGE, proteins in the gel were electrotransferred to a nitrocellulose membrane (0.45 μm, Bio-Rad, CA, USA) according to the procedure of Towbin et al. (1979). The blots were blocked with 5% non-fat dry milk in TTBS (20 mM Tris-HCl (pH 7.6), 137 mM NaCl, and 0.2% Tween-20) and then incubated with antiserum against NOS at 1:1000 dilution for 2 h. After three washes in TTBS, the blots were incubated with horseradish-peroxidaseconjugated goat anti-rabbit IgG (1:1000) in TTBS for 1 h. Immunoreactions were also carried out using actin antibody for equal protein as loading controls. Immunoreactivity was determined using the ECL chemiluminescence reaction (Amersham Bioscience, Little Chalfont, Buckinghamshire, UK) and then exposed to X-ray film. The X-ray films were scanned, and the optical densities of Western blots were analyzed by densitometry using the computer-based Sigma Gel, version 1.0 (Jandel Scientific, San Rafeal, Chicago, IL, USA).

Results cDNA cloning of H. cunea NOS and sequence characterization We obtained a partial NOS gene fragment from immunized H. cunea larvae by RT-PCR with degenerate primers based on highly conserved amino acid residues. The resulting PCR product of NOS was 960 bp. Subsequently, two fragments of 633 bp and 582 bp were amplified using specific primers. In an attempt to obtain the 5′ and 3′-ends of the coding sequence, 5′- and 3′-RACE PCRs were performed using gene specific primers. The resultant product from 3′-RACE PCR contained stop codon (TAA). However, N-terminal sequence was not obtained from 5′-RACE PCRs. A total of 1050 deduced amino acids were obtained from H. cunea NOS gene without N-terminal sequence. Multiple alignment of the predicted H. cunea NOS amino acid sequences with other known NOS sequences showed high conservation throughout the sequence with clearly identifiable putative recognition sites for co-factors and co-substrates such as heme, CaM, FMN, FAD pyrophosphate, FAD isoalloxazine, NADPH ribose, and



Table 1 Sequence members†

homology

in

insect

and

mammal

NOS

Percent identity 1

†

2

3

4

5

6

7

70.3

69.7 85.3

59.7 56.0 56.3

58.8 55.3 54.9 64.7

58.4 55.6 55.2 66.4 62.3

57.6 55.2 54.2 63.2 74.6 60.5

Identities were MEGALIGN.

determined

by

pairwise

1 2 3 4 5 6 7

HcNOS MsNOS BmNOS LcNOS AgNOS RpNOS DmNOS

alignment

using

NADPH adenine (Fig. 1), indicating that the protein is catalytically active. The H. cunea NOS is highly similar to M. sexta NOS (70.3%), Bombyx mori NOS (69.5%), Luciola cruciate NOS (59.7%), A. gambiae NOS (58.8%), R. prolixus NOS (58.4%), and D. melanogaster NOS (57.6%) (Table 1). The phylogenetic tree generated from aligned sequences of NOS and showed further that H. cunea NOS is closely related to M. sexta NOS and B. mori NOS. Reconstruction of the phylogenetic relationships based on amino acid distances revealed four sub-clades corresponding to the NOS from insects (Fig. 2). Expression profile of NOS in different tissues of H. cunea To examine the expression of NOS protein in various tissues, Western blot analysis was performed using proteins prepared from fat body, midgut, hemocytes, epidermis, and Malpighian tubules. The NOS protein was present in all tissues examined with the exception of hemocytes, indicating that NOS is ubiquitously expressed (Fig. 3). Induction of NOS protein upon bacterial infection in H. cunea In an attempt to examine the induction patterns of H. cunea NOS mRNA against bacterial infection, Western blot analysis was performed using proteins prepared from fat body, midgut, epidermis, and Malpighian tubules of last instar larvae at 1, 3, 6, 12 and 24 h intervals of challenge with E. coli or E. faecalis infection, respectively. In the time course experiment, the level of H. cunea NOS mRNA increased continuously from 3 h to 24 h in fat body of the E. coli and E. faecalis–infected larvae. In the epidermis of E. coli infected larvae, the NOS expression level increased from 6 h and peaked at 12 h but with E. faecalis

infection the increase of NOS expression began at 3 h and was found to be at a maximum at 6–12 h and then recovered at 24 h. In the midgut, the NOS expression level in both E. coli and E. faecalis infected larvae were found to be slightly increased from 6 h to 12 h and then gradually recovered to control level at 24 h post-injection. Also, in the Malpighian tubule of E. coli or E. faecalis infected larvae NOS expression level rapidly increased from 1 h to 24 h, compared with non-treated control (Fig. 4).

Discussion Nitric oxide has a number of important roles in both invertebrate (Radomski et al. 1991; Nappi et al. 2000; Foley & O’Farrell 2003) and vertebrate immunity (Abrahamsohn & Coffman 1995; Brunet 2001; Kroncke et al. 2001). The NOS produced NO by catalyzing the conversion of L-arginine to L-citrulline, with the concomitant oxidation of NADPH. The NOS gene has been cloned from various organisms, but only a few reports are available in Lepidoptera. In the present study, we identified partial cDNA encoding NOS from the fall webworm, H. cunea and investigated the induction of NOS protein in various tissues with a study on the expression profiles of NOS in H. cunea by pathogenic bacteria. Sequence analysis showed that partial cDNA of H. cunea NOS contains a polypeptide of 1050 amino acids without N-terminal sequence (about 155 amino acids) (Fig. 1). The protein sequence of NOS from H. cunea is very similar to that of NOSs from other insects (Table 1). Also, invertebrate NOS proteins contain putative recognition sites for cofactors heme, BH4, CaM, FMN, FAD, and NADPH, with the exception of H. pomatia NOS (Huang et al. 1997). These domains are very well conserved in H. cunea NOS, suggesting that conserved domains are important for the essential functions of NOS. Previous studies have demonstrated that insect NOS genes are expressed in various tissues of insects including antennal lobe, fat body, salivary gland, hemocytes, midgut and Malpighian tubule (Gibson & Nighorn 2000; Imamura et al. 2002; Whitten et al. 2007; Kraaijeveld et al. 2011). In invertebrates, NO has been implicated in several cellular, developmental, and behavioral processes (Kuzin et al. 1996; Nighorn et al. 1998; Enikolopov et al. 1999; Haase & Bicker 2003). It is also involved in the morphogenetic regulation of the nervous system (Gibbs & Truman 1998; Champlin & Truman 2000) and a signaling role in the antennal lobe and the central nervous system (Müller 1996; Nighorn et al. 1998). The ubiquitous expression of NOS supports the general observation that H. cunea NOS may play various roles of physiological functions in various tissues (Fig. 3). Figure 4 shows that the level of H. cunea NOS expression increased in the all tissues in response to either E. coli or


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Figure 1 Deduced amino acid sequence of cDNA encoding Hyphantria cunea nitric oxide synthase (NOS) and alignment with those from other insects. NOS sequences from insects were obtained from the SwissProt database and are represented as follows: Hc, Hyphantria cunea NOS; Bm, Bombyx mori NOS (GenBank accession no. NP001036963); Ms, Manduca sexta NOS (GenBank accession no. AAC61262); Rp, Rhodinus prolixus (GenBank accession no. AAB03810); Dm, Drosophila melanogaster NOS (GenBank accession no. AAF25682); Ag, Anopheles gambiae (GenBank accession no. XM31713) and Lc, Luciola cruciata (GenBank accession no. AB304920). Putative cofactor-binding sites for heme, CaM, FMN, FAD pyrophosphate (FAD-ppi), FAD isoalloxazine (FAD-Iso), NADPH ribose (NADPH-ribose), NADPH adenine (NADPH-Ade), and the C-terminal conserved sequence necessary for NADPH binding are overlined.

E. faecalis (Fig. 4). In previous reports, similar changes to NOS expression against pathogens have been reported. For example, NOS expression in fat body and hemocytes of M. sexta increased by infection of P. lumenescens, suggesting that induced synthesis of NO is important in mediating immune defense against pathogens by inhibiting transfer of bacteria across the gut wall (Eleftherianos et al. 2009). In Drosophila, inhibition of NOS increased larval sensitivity against gram-negative bacteria (Foley & O’Farrell 2003). Also in previous studies, the elevated level of NOS by immune challenge with lipopolysaccharide (LPS) increased 348

NO production, and NO is directly associated with increased diptericin expression in LPS infected tubules of Drosophila conferring increased survival of insect against pathogen (McGettigan et al. 2005). Similarly, elevated expression of NOS was detected in the midgut and carcass after invasion of the midgut by plasmodium (Luckhart et al. 1998) and in the proventriculus from immune challenged flies (Hao et al. 2003). In mosquitoes, notably Anopheles, expression of the NOS gene is upregulated in the midgut in response to infection with malaria (Dimopoulos et al. 1998; Luckhart et al. 1998; Herrera-Ortíz et al. 2004; Akman-Anderson et al.



Figure 1

Continued


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Figure 2 Distance-based phylogenetic analysis of nitric oxide synthases from Hyphantria cunea and other insects. The phenogram is based on the alignment shown in Figure 1, and distances are approximations. The names of species are described in Figure 1.

Figure 3 Expression profiles of NOS in different tissues of Hyphantria cunea. Tissues were isolated from last instar larvae. Protein (20 μg) was extracted from various tissues (20 larvae), and Western blot analysis was performed. For details see Materials and Methods. Fb, fat body; Mg, midgut; Mt, Malpighian tubule; Ep, epidermis; Hc, hemocyte.

E. Coli infection

E. faecalis infection

Trypanosoma rangeli and LPS. The most pronounced response to LPS occurred in fat body and hemocytes, while digestive tissues were most responsive to infections by T. cruzi and T. rangeli suggesting that NO-mediated immune response is pathogen-specific in R. prolixus (Whitten et al. 2007). The subsequent temporal complexity and marker specificity of the abdominal response strongly suggests that multiple organs might be induced asynchronously including the fat body and hemocytes, and additional organs (Dimopoulos et al. 1998). Nitric oxide has been implicated in playing an important role as a signal molecule in many parts of an organism as well as a cytotoxic effector molecule of the non-specific immune response (Krishnan et al. 2006). Our result suggests that the H. cunea NOS could play an important role in the defense system against bacterial infection in different tissues in a time-specific manner.

Acknowledgments

Figure 4 Expression patterns of NOS from the last instar larvae of Hyphantria cunea in response to Gram negative (Esherchia coli) and Gram positive (Enterococcus faecalis) bacterial injections. Protein (20 μg) was extracted from the various tissues (20 larvae) at 1 h, 3 h, 6 h, 12 h and 24 h after injection, respectively. Western blot analysis was performed. For details, see Materials and Methods. Con, salineinjected control; Fb, fat body; Mg, midgut; Mt, Malpighian tubule; Ep, epidermis; At, Actin (loading control).

2007). (Their observations demonstrated that reactive oxygen and nitrogen intermediates constitute a part of cytotoxic arsenal employed by Anopheles mosquitoes against microbial pathogens and plasmodium ookinetes (Herrera-Ortíz et al. 2004). Tissue and time-specific alteration in NOS gene expression was documented in R. prolixus following in vivo immune challenge by Trypanosoma cruzi, 350

This work was supported by the Korea Science and Engineering Foundation (KOSEP) and the National Research Foundation of Korea (grant no. 2009-0067292). HJ Kim was supported by the National Research Foundation of Korea (grant no. 359-2008-1-C00035).

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