Antimicrobial peptide gene cecropin-2 and defensin

Comparative Biochemistry and Physiology, Part B 206 (2017) 1–7

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Antimicrobial peptide gene cecropin-2 and defensin respond to peptidoglycan infection in the female adult of oriental fruit fly, Bactrocera dorsalis (Hendel) Shi-Huo Liu, Dong Wei, Guo-Rui Yuan, Hong-Bo Jiang, Wei Dou, Jin-Jun Wang ⁎ Key Laboratory of Entomology and Pest Control Engineering, College of Plant Protection, Southwest University, Chongqing 400716, PR China

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Article history: Received 14 November 2016 Received in revised form 6 January 2017 Accepted 10 January 2017 Available online 12 January 2017 Keywords: Antimicrobial peptide Cecropin-2 Defensin Bactrocera dorsalis Immunity

a b s t r a c t Cecropins and defensins are important antimicrobial peptides in insects and are inducible after injection of immune triggers. In this study, we cloned the cDNAs of two antimicrobial peptides (AMPs), cecropin-2 (BdCec-2) and defensin (BdDef) from Bactrocera dorsalis (Hendel), a serious pest causing great economic losses to fruits and vegetables. The BdCec-2 sequence of 192 bp encodes a protein of 63 amino acids residues with a predicted molecular weight of 6.78 kD. The 282 bp cDNA of BdDef encodes a protein of 93 residues with a predicted molecular weight of 9.81 kD. Quantitative real-time PCR analyses showed that BdCec-2 and BdDef had similar expression profiles among development stages, the highest mRNA levels of these two AMP genes were observed in the adult stage. Among different adult body segments and tissues, both genes had similar transcriptional profiles, the highest mRNA levels appeared in abdomen and fat body, which was consistent with the reported fact that fat body was the main organ synthesizing AMPs in insects. The expression of BdCec-2 and BdDef were up-regulated after challenge with peptidoglycans from Escherichia coli (PGN-EB) and Staphylococcus aureus (PGN-SA), respectively, suggesting their antimicrobial activity against Gram-negative and Gram-positive microorganisms. These results describe for the first time the basic properties of the cecropin-2 and defensin genes from B. dorsalis that probably play an important role in the defense response against invading microbes. © 2017 Elsevier Inc. All rights reserved.

1. Introduction Because of the disability of insect to synthesize antibodies in response to immune challenge, insect innate immunity lacks specificity and memory (Schmid-Hempel, 2005). To defend against microbial infection, insects have possessed a large range of strategies based on cellular and humoral immune mechanisms. Antimicrobial peptides (AMPs) are the main factors of humoral responses and are synthesized mainly in the insect fat body (Hultmark, 1993; Hoffmann, 1995). AMPs have been the focus of intense research about immunity in the past decades, and now they are considered as an essential part of the defense system in insects. The immune-inducible AMPs produced by D. melanogaster have been identified and were grouped into eight classes: cecropins, attacins, defensins, drosocins, diptericins, drosomycins, metchnikowin and bomanin (Lindmark et al., 2001; Roxstrom-Lindquist et al., 2004; Lemaitre and Hoffmann, 2007; Clemmons et al., 2015). Sometimes, lysozymes are considered as AMPs, in fact, this topic is controversial, some researchers recommend lysozymes as a digestive molecular ⁎ Corresponding author at: College of Plant Protection, Southwest University, Chongqing 400715, PR China. E-mail address: [email protected] (J.-J. Wang).

http://dx.doi.org/10.1016/j.cbpb.2017.01.004 1096-4959/© 2017 Elsevier Inc. All rights reserved.

which are commonly released into the gut to digest microflora (Lemaitre and Hoffmann, 2007). In Bombyx mori, four categories of common AMPs (cecropins, attacins, defensins, and lysozymes) and three categories of Lepidopteran insects unique AMPs including moricins, lebocin and gloverin, were identified, respectively (Abraham et al., 1995; Kaneko et al., 2007). Among these AMPs, the cecropins and defensins are particularly important because of their diverse functions. The cecropin was the first identified AMP from Hyalophora cecropia (Steiner et al., 1981). Subsequently, it has been widely found in insects and plays a crucial role in insect immune system. Cecropin is also one of the most functionally diverse antimicrobial peptides known, exhibiting a wide spectrum of antibacterial activity against Gram-positive and Gram-negative bacteria as well as viruses (Imamura et al., 2006; Jan et al., 2010). In contrast, the cysteine-rich polypeptide defensin was first detected in the flesh fly, Sarcophaga peregrine (Matsuyama and Natori, 1988). Thereafter, over 70 different defensins have been isolated from various invertebrates such as insects, crustaceans, ticks, spiders, scorpions, and mollusks (Bulet et al., 2004). Defensins are the largest family of AMPs and are ubiquitous in almost all life forms including animals, fungi and plants. They possess broad-spectrum antimicrobial activity against bacteria, fungi and enveloped viruses. Antimicrobial peptides are expressed constitutively or induced after microbial infection (Lemaitre and Hoffmann, 2007). As reported, AMPs

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S.-H. Liu et al. / Comparative Biochemistry and Physiology, Part B 206 (2017) 1–7

are mainly produced by the activation of the insect fat body, and released into hemolymph (Hoffmann et al., 1999). Otherwise, the induction of different insect AMPs, as well as induction in different tissues including fatbody, reproductive tissues, midgut and salivary gland in various insects has been reported (Tzou et al., 2002; Wang et al., 2013b; Liao et al., 2015). The oriental fruit fly, Bactrocera dorsalis (Hendel), is one of the most economically important fruit fly pests in East Asia and the Pacific (Wang et al., 2013a). It was first recorded in Taiwan, China in 1912 (Hardy, 1973; Drew and Hancock, 1994) and expanded its range to Hainan Island in 1934 (Xie, 1937). By 2003, this species was reported from the Chinese provinces of Guangdong, Fujian, Guangxi, Hainan, Yunnan, Guizhou, Sichuan, Hunan and Taiwan (Liang et al., 2002). Recently, the oriental fruit fly has spread north-eastward into Chinese provinces of Jiangsu, Zhejiang and Shanghai (Zhou et al., 2006; Qi et al., 2008; Zhao et al., 2008). To date, only four groups of AMPs have been identified in this species: cecropins, attacins, bactrocerin and diptericins (Dang et al., 2009; Jiang et al., 2014; Liao et al., 2015; Hanson et al., 2016). To better understand the adaption of B. dorsalis to new environment, it is necessary for us to identify more AMPs genes which may contribute to its expansion by enhancing immune system from B. dorsalis and analyzes the immune response challenged by some immune triggers. In the present study, we first identified a novel B. dorsalis cecropinencoding gene (BdCec-2) and a defensin-encoding gene (BdDef), which have significant homology with other insect cecropins and defensins, respectively. Subsequently, the temporal and developmental expression profiles of genes were investigated. Finally, we determined the mRNA expression patterns of BdCec-2 and BdDef in female adults in response to peptidoglycan immune challenge. The results of this study provide information that may be helpful in guiding biological control of B. dorsalis with microorganism. 2. Materials and methods 2.1. Insects The laboratory colony of B. dorsalis was originally collected in 2008 from Hainan province of China, and maintained in the insectary at 27.5 ± 0.5 °C, 75 ± 5% relative humidity (RH) and a photoperiod of 14:10 (L:D). Larvae and adults were fed with artificial diet described previously (Wang et al., 2013a). During experiment, female adults and male adults were separated immediately after eclosion and were raised in different cages to avoid mating before being collected. 2.2. Total RNA extraction and cDNA synthesis Total RNA of each sample was extracted using TRIzol® reagent (Invitrogen, Carlsbad, CA, US). The quality and concentration of RNA were measured by NanoVue UV–Vis spectrophotometer (GE Healthcare Bio-sciences, Uppsala, Sweden), and the integrity of isolated RNA was checked by 1.0% agarose gel electrophoresis. Prior to cDNA synthesis, total RNA was treated with RQ1 RNase-Free DNase (Promega, Madison, WI, USA) to digest genomic DNA. For first strand cDNA synthesis, 500 ng of total RNA was reverse transcribed using the PrimeScript 1st Strand cDNA Synthesis Kit (Takara, Dalian, China) with random hexamers and oligo (dT) primers, according to the manufacturer's instructions. 2.3. Molecular cloning Based on transcriptomic data of B. dorsalis (NCBI SRA accession number: SRR1168415), the specific primers for BdCec-2 (forward, 5′GGAAGCACACCAAGTGTAATATCT-3′; reverse, 5′-CTTATCCCTTCAAT GTTGCTGC-3′) and BdDef (forward, 5′-ACCCACTGAAGGAGATAGCG-3′; reverse, 5′-CGAGAACCACGCAATGACTT-3′) were designed using primer premier 5.0 (Premier Biosoft International, Palo Alto, CA, USA) to amplify the ORFs of both AMPs genes, respectively. PCR parameters were

94 °C for 3 min; 35 cycles of 94 °C for 30 s, anneal for 30 s, and extend at 72 °C for 1 min; 72 °C for 7 min. All PCR products were cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA) and sequenced by Invitrogen in Shanghai, China. 2.4. Sequence analysis and phylogenetic tree construction The ExPASy Proteomics Server (http://cn.expasy.org/tools/pi_tool. html) was used to compute the isoelectric point and molecular weight of the deduced protein sequences. The signal peptides were predicted by the SignalP 4.1 server program (http://www.cbs.dtu.dk/services/ SignalP). Multiple sequence alignments were performed with online software MAFFT version 7 (http://mafft.cbrc.jp/alignment/server/). The α-helix structures of the BdCec-2 were predicted by SOPMA secondary structure prediction method (https://npsa-prabi.ibcp.fr/cgibin/npsa_automat.pl?page=/NPSA/npsa_sopma.html) (Combet et al., 2000). The cysteine disulfide bonding state and connectivity of BdDef was predicted by the DISULFIND (http://disulfind.dsi.unifi.it/). The phylogenetic trees were constructed on the basis of amino acid sequences by the software MEGA6.0 (Tamura et al., 2011) using the Maximum Likelihood (ML) method, respectively. Bootstrap values were calculated with 1000 replications. The 24 amino acid sequences for phylogenetic tree construction of cecropins were come from Drosophila, Bactrocera, Musca, Trichoplusia, Hyalophora, Bombyx and Hyphantria. The 17 amino acid sequences for phylogenetic tree construction of defensins were come from Drosophila, Bactrocera, Copris, Oryctes, Anomala, Trachymyrmex, Apis and Polistes. All sequences were downloaded from the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov). 2.5. Gene expression analysis Insects at different developmental stages including eggs, 3rd instar larvae, 5-day-old pupae and 5-day-old adults were collected, respectively, the weight of each sample was about 50 mg. Each sample was collected in triplicates, immediately frozen in liquid nitrogen, and stored at −80 °C for further use. To obtain different body parts, 5-day-old female and male adults were used. Six individual of adults were dissected for head, thorax and abdomen using operating scissor. Each sample was collected in triplicates, immediately frozen in liquid nitrogen, and stored at − 80 °C for RNA isolation. In the tissue-specific experiment, 3 to 5-day-old female and male adults (no mating before being collected) were used for tissue isolation, respectively. The midgut, Malpighian tubules, and fat body of B. dorsalis adults were dissected under a stereomicroscope (Olympus SZX12, Tokyo, Japan) and placed in a 1.5 mL centrifuge tube containing RNA store reagent (Tiangen, Beijing, China). Twenty individual of adults were dissected for midgut and fat body tissue, and forty adults were used for Malpighian tubules. All tissue sample were stored at − 80 °C for RNA isolation. Three biological replicates were performed. Quantitative real-time PCR (qRT-PCR) was performed to detect the relative expression of BdCec-2 and BdDef during various developmental stages, different body parts and different tissues of the oriental fruit fly, as well as female adults treated by immune triggers. The qRT-PCR was performed using Mx3000P thermal cycler (Stratagene, La Jolla, CA, USA) with the stable B. dorsalis reference gene α-tubulin (GU269902) (Shen et al., 2012). All qRT-PCR were carried out in 20 μL reaction containing 1 μL template cDNA (600 ng/μL), 10 μL iQTM SYBR Green Supermix (Bio-Rad, Hercules, CA, USA), 1 μL each of forward and reverse primers (10 μM), and 7 μL nuclease-free H2O. Primers for BdCec-2 (forward, 5′-TTCGTTCTCCTGGCTGTCTT-3′; reverse, 5′-ATCCCTTCAATGT TGCTGCC-3′) and BdDef (forward, 5′-ACCCACTGAAGGAGATAGCG-3′; reverse, 5′-CGAGAACCACGCAATGACTT-3′) and for the internal reference gene α-tubulin (forward, 5′-CGCATTCATGGTTGATAACG-3′; reverse, 5′-GGGCACCAAGTTAGTCTGGA-3′) were designed using online


software Primer 3 Web (http://primer3.ut.ee/). The reaction conditions were: 95 °C for 2 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 30 s. After the cycling protocol, the melting curve analysis from 60 °C to 95 °C was used to verify a single PCR product. The relative mRNA levels were calculated according to the 2−ΔΔCt method (Pfaffl, 2001). The expression data were expressed as means ± standard error (SE). The statistical analysis was performed using SPSS 19.0 (IBM, Chicago, IL, USA).

2.6. Immune challenge Peptidoglycan from Staphylococcus aureus (PGN-SA, InvivoGen, San Diego, CA, USA) and Escherichia coli 0111:B4 (PGN-EB, InvivoGen) were diluted into sterile endotoxin-free water (additional contents in the packaging of PGN, InvivoGen), respectively, at a final concentration of 100 μg/mL. Five-day-old female adult flies were injected with 200 nL PGN-SA solution (PGN-SA group), PGN-EB solution (PGN-EB group) and sterile endotoxin-free water (water group), respectively. The injury group was just pricked with the microinjection needle without injection, and the blank group was neither injected nor pricked. Three individual of female adult flies were randomly collected from each group after 3, 6 and 9 h post injection. Each treatment was performed in triplicates. All samples were stored at −80 °C for RNA isolation.

3. Results 3.1. Sequence and phylogenetic analysis of BdCec-2 and BdDef The full-length of open reading frame (ORF) of BdCec-2 (GenBank accession no. KX510001) and BdDef (GenBank accession no. KX510002) cDNAs were cloned, respectively. The length of ORF of BdCec-2 and BdDef were 192 bp and 282 bp, respectively. The 62 amino acid residues encoded by BdCec-2 and 93 amino acid residues encoded by BdDef constituted BdCec-2 peptide and BdDef peptide, respectively. The predicted molecular mass and isoelectric point of BdCec-2 peptide and BdDef peptide were 6.78 kDa, 10.29 and 9.79 kDa, 8.90, respectively. The first 23 amino acids of BdCec-2 and the first 22 amino acids of BdDef at the N-terminus were predicted as signal peptide (Figs. 1 and 2). Two α-helix regions were predicted at the N and C termini of mature BdCec-2 after the excision of the signal peptide (Fig. 1). And three cysteine disulfide bonds were predicted at the mature peptide region of BdDef (Fig. 2). The phylogenetic analysis showed that Cecropins in Diptera and Lepidoptera species were separated into two corresponding clades (Fig. 3). The BdCec-2 is closely related to the olive fruit fly cec-1-like with a bootstrap value of 68%. Meanwhile, the phylogenetic analysis showed that Defensins in Diptera, Coleoptera and Hymenoptera species were separated into three corresponding clades (Fig. 4). The BdDef is closely related to the olive fruit fly Def-like with a bootstrap value of 55%.

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3.2. Temporal and developmental expression profiles of BdCec-2 and BdDef As shown in Fig. 5A, the developmental expression profiles revealed that the highest and lowest mRNA levels of BdCec-2 existed in adult and egg, respectively. The transcriptional mRNA level of BdCec-2 in the egg stage was negligible compared to adult stage. In the different adult body parts, the highest mRNA level was observed in the abdomen compared with the head and thorax in female and male (Fig. 6A). In the different abdominal tissues, the highest mRNA level was observed in the fat body compared with the Malpighian tubule and mid gut, regardless of the sex of insect (Fig. 6C). Similar to the expression profiles of BdCec-2, the highest and lowest mRNA levels of BdDef were also in adult and egg, respectively (Fig. 5B). The transcription profiles of BdDef in the body parts and abdominal tissues were similar to those of BdCec-2. In the different adult body parts, the highest mRNA level of BdDef was observed in the abdomen compared with the head and thorax in female and male (Fig. 6B). As for the mRNA level of BdDef in a certain body part among head, thorax and abdomen, female significantly higher than that of male, In the different abdominal tissues, the highest mRNA level was observed in the fat body compared with the Malpighian tubule and mid gut, regardless of the sex of insect (Fig. 6D). 3.3. Transcriptional response of BdCec-2 and BdDef to immune challenge As shown in Fig. 7, the relative mRNA levels of BdCec-2 significantly increased 9.98- and 7.32-fold at 3 h, and 9 h after PGN-EB injection, comparing to mRNA levels after water injection (Fig. 7A). Although the BdCec-2 was 12.91-fold up-regulated compared to blank group at 6 h post injection, the relative mRNA level of BdCec-2 after PGN-EB injection was only 1.39-fold up-regulated compared to that of water injection, and there is no significant difference between water treated group and PGN-EB treated group (Fig. 7A). The injection of PGN-SA did not show significantly induction of BdCec-2 at 3 h and 6 h after injection, except 9 h post injection (5.32-fold comparing to the water group) (Fig. 7A). The relative mRNA levels of BdDef were significantly increased at 3 h, 6 h and 9 h after injected PGN-EB, and the ratios of inducible expression to constitutive expression of BdDef were 2.30-, 2.21- and 2.32- fold (compared to mRNA levels of water injection) at 3 h, 6 h and 9 h post treatment (Fig. 7B). The injection of PGN-SA could not significantly induce BdDef at 3 h and 9 h after injection, however, BdDef RNA level was significantly increased at 6 h post injection (Fig. 7B). 4. Discussion To date, there are N2600 antimicrobial peptides that have been collected on the Antimicrobial Peptide Database (APD: http://aps.unmc. edu/AP/main.php). However, insect AMPs only consist of b10% of them, and thus studies on antimicrobial peptides of insects show great importance to our knowledge and application of insect AMPs. In addition, recent researches show that AMP polymorphism of Drosophila

Fig. 1. Sequence alignments of BdCec-2 with other insect cecropins. The amino acid sequence of BdCec-2 is aligned with those of the Bactrocera dorsalis (BdCec, AJF11662; BdCec-2, KX510001); Drosophila melanogaster (DmCecB, AAB82493), Drosophila simulans (DsCecB, KMZ06808), Drosophila erecta (DeCecC, KQS52037) and Drosophila virilis (DvCec2B, EDW59216). The signal peptide of BdCec-2 is on a gray background. Amino acids corresponding to predicted α-helix are underlined. Identical residues are indicated by * under the residues.

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Fig. 2. Multiple sequence alignment of amino acid sequence of BdDef with that of other insect defensins. The amino acid sequences are from the oriental fruit fly, B. dorsalis (BdDef, KX510002), Bactrocera oleae (BoDef, XP_014095036); the western honey bee, Apis mellifera (AmDef, AAS75803); the cupreous chafer, Anomala cuprea (AcDef, BAD77967); and the paper wasp, Polistes dominula, (PdDef, ADB85559). The signal peptide of BdDef is on a gray background. The conserved mature peptide cleavage sites, KR or RR, are boxed, and the mature peptide of BdDef is underlined. Identical residues are indicated by * under the residues. The triangle indicates the six conserved cysteine residues. And the dashed line indicates the disulfide bond.

affects bacterial resistance of host (Unckless et al., 2016), which inspires us to perform species control by applying AMPs. Cecropins are a family of cationic AMPs with 58–64 amino acids and have been identified in dipteran, lepidopteran, and coleopteran insects. After the removal of the signal peptide, the mature cecropins form two α-helixes connected by a hinge and the N-terminal α-helix is presumptively required for the biological activity of cecropins (Wang et al., 2008). Multiple sequence alignments revealed that BdCec-2 shared

strong conservation of the mature peptide sequence with those from other insects. The defensin family is a group of ubiquitous AMPs found in insects. The insect defensin usually consists of a signal peptide region, prodefensin and the mature peptide. As previously reported, mature peptide and prodefensin were cleaved at the ‘KR’ cleavage site (Lowenberger et al., 1999), however, it was also present as ‘RR’ in BdDef in this study (Fig. 2). Multiple sequence alignments found that

Fig. 3. Phylogenetic analysis of 24 cecropins from insect. The phylogram of 24 insect cecropin amino acid sequences was generated in MEGA6.0 using Maximum Likelihood method. Bootstrapping analysis was performed 1000 replications. Accession numbers were labeled together with scientific name.


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Fig. 4. Phylogenetic analysis of 17 defensins from insect. The phylogram of 17 insect defensin amino acid sequences was generated in MEGA6.0 with Maximum Likelihood method. Bootstrapping analysis was performed 1000 replications. Accession numbers were labeled together with scientific name.

BdDef shared strong conservation of the mature peptide sequence with those from other insects, particularly the location of six cysteine residues (Fig. 2), which form three disulfide bridges. These three disulphides adopted a ‘cysteine stabilized αβ’ (CS αβ) scaffold (Cornet et al., 1995). The predicted result of cysteine disulfide bonding state and connectivity showed that the pairing patterns of these six cysteine residues were Cys1–Cys4, Cys2–Cys5 and Cys3–Cys6. It is most likely that such conservation construction exists due to the importance of defensins as key components in insect immune system that fight against invaded pathogens. Studies on AMPs demonstrated that antimicrobial activities against diverse microbes were associated with net positive charges (Yeung et al., 2011; Wang et al., 2013b). We found that mature peptide of BdCec-2 and BdDef to have these charges at pH 7.0: +5.1 and +4.8, respectively. Because of an excess of positive charges contained by BdCec2 and BdDef, they may easily react with microbial membranes. AMPs are mainly produced in insect fat body and released into the hemolymph, which will in turn induce other immune effectors. They can also be delivered to other tissues (Hoffmann et al., 1999). Although different insects are capable to produce AMPs belonging to the same family, the mRNA level and the function of specific AMPs differ a lot among species, families and orders of insect. AMPs may be differentially expressed in various developmental stages, different body parts and tissues depending on insect species. The developmental expression pattern of cecropins was reported in B. dorsalis (BdCec) (Liao et al., 2015) and Anopheles gambiae (AngCec) (Vizioli et al., 2000) without any immunization. In a previous staudy,

BdCec was highly expressed in the first-instar larvae and later third-instar larvae, while it was very lowly expressed at the pupal stage. On the contrary, AngCec was significantly expressed in early pupae but not in larval stages. This suggested that cecropin peptide probably play important roles in fighting against invaded microorganisms during the firstinstar larvae in B. dorsalis and the early pupa stage in A. gambiae. However, our results on BdCec-2 were different from both cases. BdCec-2 was highly expressed at adult stage, and relative lowly expressed in larvae and pupae. Although BdCec and BdCec-2 belong to the same cecropin family, the different expression profiles of them probably suggest their different function involvement in immune system. The expression profiles of defensins were reported in Bombyx mori (Wen et al., 2009) and Drosophila melanogaster (Dimarcq et al., 1994). A high level of constitutive expression of BmDefA was observed in early pupae and early moth, similar to BmDefA, the defensin gene of D. melanogaster was expressed spontaneously in pupa and adult with no immune challenge. However, BdDef was highly expressed at the larva and adult stages (Fig. 5). This suggested that BdDef played important roles in fighting against invaded microbes in larva and adult stages. Previous studies showed that transcripts of most insect AMPs are primarily distributed in immune-related organs such as the fat body, midgut or haemolymph (Wang et al., 2006). Furthermore, it has been well documented that the expression profiles of AMPs in different tissues depend on the category of AMP gene with variations across insect species. In Bemisia tabaci, BtDef was expressed in all four tissues including fat body, midgut, salivary gland and ovary (Wang et al., 2013b). Among the tissues, BtDef was highly expressed in midgut. In contrast,

Fig. 5. The relative mRNA expression profiles of BdCec-2 and BdDef during different developmental stages of B. dorsalis. The expression profiles of BdCec-2 and BdDef during egg, the third instar larva, 5-day-old pupa and 5-day-old adult. Data are presented as mean ± SE (n = 3). Different letters above each bar indicate statistical difference by ANOVA followed by the Tukey's Multiple Comparison test (P b 0.05).

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Fig. 6. The relative mRNA expression profiles of BdCec-2 and BdDef in female and male's body parts and tissues of B. dorsalis. Expression profiles from female and male adult body parts including the head, thorax and abdomen, and the tissues including malpighian tubule (MT), mid gut (MG) and fat body (FB). Data are presented as mean ± SE (n = 3). The twotailed, unpaired t-test was used to test significance. Asterisks indicate significant differences in relative expression. (*, p b 0.05; **, p b 0.01).

there was no expression of BmDefA in the midgut of B. mori (Wen et al., 2009). In the current study, both BdCec-2 and BdDef showed highest expression in fat body. Thus, we speculate that both BdCec-2 and BdDef undertake their roles mainly in the fat body of adults to protect B. dorsalis from pathogens infection. Although BdCec-2 and BdDef do not belong to the same family, the similar expression pattern in body parts and tissues may be necessary for B. dorsalis in response to infection with different microbes (Figs. 6 and 7). To further study the immune response of insects, we may choose to inject microbes into insects. However, injection of the live microbes leads to complex immune responses because of potential growth of the microbe within the host. Although injection of dead bacteria allows circumventing this problem, some uncertain component of the injected bacteria may cause immune responses. In order to activate a specific branch of the immune response pure elicitors can be used such as pure products of peptidoglycan (PGN) from gram-positive and gramnegative bacterium (Neyen et al., 2014).

Though there was some AMPs failed to be induced in Drosophila (Hanson et al., 2016), injection of PGN to induce AMPs expression has been considered as a good way to investigate insect immune response. The time courses (3, 6, and 9 h post injection of PGN) of transcriptional responses of BdCec-2 and BdDef were determined in the females. Transcription levels of BdDef were significantly up-regulated at 3, 6 and 9 h after PGN-EB challenge, while injection of PGN-SA only induced the expression of BdDef 6 h post the injection. This indicated that the BdDef could be consecutively induced by Gram-negative PGN but transiently induced by Gram-positive PGN. The pattern of induction of BdDef is similar with that of Drosophila (Lemaitre et al., 1997). The transcription of the Drosophila defensin was marked by a strong induction by Gramnegative bacterium (E. coli) at 3 h, and a weak response by Gram-positive bacterium (M. luteus) at 3, 6 and 12 h after challenge. On the other hand, there was some difference between the induction of BdDef by PGN-EB and the induction of Drosophila defensin gene by E. coli. For instance, infection of E. coli only weakly induced Drosophila defensin at 6 h

Fig. 7. Effects of PGN-EB and PGN-SA challenge on the mRNA levels of BdCec-2 and BdDef. Relative mRNA levels of BdCec-2 and BdDef after PGN-EB and PGN-SA challenge. Blank: neither injected nor pricked; Injury: just pricked with the microinjection needle without injection; Water: injected with 200 nL sterile endotoxin-free water; PGN-EB: injected with 200 nL PGN-EB solution; PGN-SA: injected with 200 nL PGN-SA solution. All values were normalized relative to the blank group (set as 1). Data represent mean 2−ΔΔCt values of three independent biological replicates ± SE. The two-tailed, unpaired t-test was used to test significance. Asterisks indicate significant differences in relative expression. (*, p b 0.05; **, p b 0.01).


after challenge, but injection of PGN-EB could strongly induced BdDef at 6 h post challenge. As for this difference, it might be due to different responses of innate immune system to E. coli and PGN-EB. Moreover, BdCec-2 showed significantly different response to the injection of PGN-EB. BdCec-2 was significantly induced at 3 post challenge, but its expression decreased gradually from 3 h to 9 h. It is interesting that PGN-SA significantly up-regulated BdCec-2 at 9 h post injection with a 5.32-fold (compared with water group). A similar phenomenon was found in Bombyx mori (Hong et al., 2008), the Cecropin-D and Cecropin-E genes of Bombyx mori were strongly induced by E.coli (Gram-negative) but weakly induced by M. luteus (Gram-positive). This suggested that BdCec-2 responses to Gram-negative microorganisms sensitively, but bluntly to Gram-positive microbes. In summary, we firstly cloned and characterized defensin gene and cecropin-2 gene from B. dorsalis, and investigated the spatiotemporal expression patterns of these two AMP genes. Furthermore, we determined the mRNA expression profiles of BdCec-2 and BdDef in female adults in response to peptidoglycan immune challenge, which is an important step and lays the foundation for understanding functional properties, and the molecular mechanisms in immune system. However, further studies such as RNAi and heterologous expression is needed to elucidate the exact function of these AMPs in response to microorganism invasion and potential contribution to the adaption to new environment causing the expansion of B. dorsalis. Meanwhile, more AMPs and other effector molecules of innate immunity of the B. dorsalis are expected to be mined to determine how the oriental fruit fly perceives infection and fights against invaded pathogens, which will benefit the species control and extension the source of novel antibiotics (Mylonakis et al., 2016) in healthcare and applying AMPs as bactericide in agriculture fields.

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