Antimicrobial peptide gene BdPho responds to peptidoglycan infection and mating stimulation in oriental fruit fly, Bactrocera dorsalis (Hendel)

Phormicins belong to defensin family, which are important antimicrobial peptides (AMPs) in insects. These AMPs are inducible upon challenging by immune triggers. In the present study, we identified the cDNA of a phormicin gene (BdPho) in the oriental fruit fly, Bactrocera dorsalis (Hendel), a ruinous agricultural pest causing great economic losses to fruits and vegetables. The cDNA of BdPho contains a 282 bp open reading frame encoding 93 amino acid residues, and the predicted molecular weight and isoelectric point of BdPho peptide were 9.83 kDa and 7.54, respectively. Quantitative real-time PCR analyses showed that the transcription level of BdPho was the highest in adult during different developmental stages and was the highest in abdomen among adult tagmata. Moreover, BdPho was highly expressed in fat body among different tissues, both in female and male adult. The mRNA level of BdPho was significantly up-regulated to 7.46- and 14.53-fold at 3 and 6 h after the insects were challenged with peptidoglycans from Escherichia coli (PGN-EB), respectively, suggesting its antimicrobial activity against Gram-negative microorganisms. Furthermore, the expression level of BdPho was significantly up-regulated to 3.83-fold after mating, suggesting that female adults might enhance their immunity by up-regulating the expression level of BdPho during mating. These results firstly describe the basic properties of the phormicin gene from B. dorsalis, and lay the foundation for investigating functional properties of AMPs and exploring the molecular mechanisms in the immune system. Electronic supplementary material The online version of this article (10.1186/s13568-017-0533-8) contains supplementary material, which is available to authorized users.


Introduction
The natural immune system of insect consists of cellular and humoral responses. The cell-mediated immunity includes phagocytosis, pinocytosis, nodulation, and encapsulation of invading microbes (Schmidt et al. 2001). In contrast, the humoral response includes melanization and the secretion of antimicrobial peptides (AMPs). AMPs are one of the most essential molecules of the humoral immune response and are synthesized mainly in insect fat body (Hoffmann 1995;Hultmark 1993). During the past decades, AMPs have been the focus of intense research on insect immunity and are now regarded as a vital part of the defense system in insects. Insect AMPs are not only involved in the defense against invading microorganisms in the haemolymph, but also regulate the bacterial community homeostasis in host gut (Buchon et al. 2013). It has been reported that the categories and expression levels of AMP genes could affect bacterial resistance of host in Drosophila (Unckless et al. 2016) and gut symbiont which could in turn enhance insecticide resistance in Bactrocera dorsalis (Cheng et al. 2017). These interesting results broadened our knowledge in pest control and showed that insect AMPs could serve as target or gut-symbiont-killer to control pest indirectly.
Defensins are the largest family of AMPs and are found in almost all organisms including animals, plants and microorganisms. The cysteine-rich polypeptide defensin was first isolated from the flesh fly, Sarcophaga peregrine (Matsuyama and Natori 1988). Thereafter, more and more different defensins have been identified from various insects. Insect defensins are widely investigated in the orders of Diptera, Coleoptera, Lepidoptera, Hemiptera, and Hymenoptera (Liu et al. 2017;Tian et al. 2010;Ueda et al. 2005;Wang et al. 2013b;Wen et al. 2009). Note worthily, defensin is also present in the ancient order of Odonata (Bulet et al. 1992), suggesting that insect defensins may derive from a common ancestor gene. Phormicin (also called phormia defensin) is a cysteine-rich antimicrobial peptide belonging to the defensin family. It was firstly identified in Phormia terraenovae (Lambert et al. 1989). Phormicins have broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria, as well as fungi (Lambert et al. 1989;Lamberty et al. 1999Lamberty et al. , 2001. The oriental fruit fly, B. dorsalis (Hendel) belonging to Tephritidae of Diptera, is one of the most destructive agricultural pests in many regions of the world (Clarke et al. 2005). Dipteran insects are considered as microorganism-rich and expected to be abundant in AMPs. To date, only five groups of AMPs have been identified in this species, including cecropins, defensins, attacins, bactrocerin, and diptericins (Dang et al. 2009;Jiang et al. 2014;Liao et al. 2015;Liu et al. 2017). To better understand the adaptation of B. dorsalis to different environments and also provide more natural molecules with potential for designing effective antibiotics applied in medicine and agriculture (Yi et al. 2014), it is worthy to identify more AMPs and analyze the immune response challenged by the immune triggers in B. dorsalis. Therefore, we initiated this study to identify a phormicinencoding gene (BdPho) in B. dorsalis, and to investigate the response model of BdPho to immune-stimulation. The results of this study provided fundamental information for understanding the molecular mechanisms in immune system of Tephritidae insects, and also provide innovative ideas for developing effective insecticides.

Insects
The oriental fruit fly, B. dorsalis was originally obtained from Hainan province of China in 2008, and maintained under laboratory conditions at 27.5 ± 0.5 °C, 75 ± 5% relative humidity (RH), and a photoperiod of 14:10 (L:D). Larvae and adults were reared on artificial diets as previously described (Wang et al. 2013a). To obtain virgin adults, newly enclosed adults were sex-biased separated and reared in different cages until collection.
For the spatiotemporal expression profiles of BdPho, samples at different developmental stages and different tissues of adult were collected as described previously (Liu et al. 2017).

RNA isolation and cDNA synthesis
Total RNA of each sample was isolated separately using TRIZOL reagent (Invitrogen, Carlsbad, CA) according to the manufacturer instructions. Before cDNA synthesis, the contaminating genomic DNA of each RNA sample was digested with DNase I (Promega, Madison, WI). First-strand cDNA was synthesized as described previously (Liu et al. 2017).

Molecular cloning
The specific primers used for amplifying open reading frame of BdPho (forward, 5′-TTTCACACACCTCAGTA AACTTTCT-3′; reverse, 5′-TTATTCCCCTCTCTTCAG TCGT-3′) were synthesized by Invitrogen in Shanghai, China. All PCRs were performed with 25 μL reaction mixtures as described previously (Liu et al. 2017). PCR parameters were: 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, anneal of 58 °C for 30 s, and extension of 72 °C for 1 min, final extension of 72 °C for 7 min. The purified PCR products were sequenced by Invitrogen.
The phylogenetic analysis on the basis of amino acid sequences was performed with MEGA 6.0 (Tamura et al. 2011)

Immune challenge
The purified peptidoglycan from the Gram-positive bacterium Staphylococcus aureus (PGN-SA, InvivoGen, San Diego, CA) and Gram-negative bacterium E. coli O111:B4 (PGN-EB, InvivoGen) were used as immune triggers. PGN-EB and PGN-SA powder were diluted with the sterilized endotoxin-free water (InvivoGen) to a final concentration of 100 μg/mL, respectively. Eighty 5-dayold virgin female adult flies were randomly collected and divided into four groups: named PGN-EB group, PGN-SA group, control group, and blank group, respectively. Each adult of PGN-EB group, PGN-SA group, and control group were injected with 200 nL PGN-EB solution, PGN-SA solution, and the sterilized endotoxin-free water, respectively, and no injection for adults in blank group. Three individuals of flies were randomly collected from each group after 3, 6 and 9 h post injection, respectively. Experiments were repeated three times independently and total RNA of each sample was extracted as described above.

Mating experiment
Female and male adults were sex-biased reared for 9 days after eclosion, respectively. On the 10th day, 20 individuals of each female and male flies randomly collected from corresponding cage and matched in a new cage to allow mating at dark. When female and male flies begin mating, the couples were carefully transferred to small cages and raised for 12 h, avoiding disturbing. Finally, six individuals of each mated and virgin female flies were randomly collected. Total RNA isolation was performed as described above. The experiment was repeated three times independently.

Data analysis
The relative expression levels were calculated based on the expression level of reference genes by qBase (Hellemans et al. 2007) and data were analyzed with SPSS 19.0 software (IBM, Chicago, IL). Significant differences were analyzed with t-test and one-way ANOVA (followed by a Tukey test) for comparison of two means or multiple comparisons, respectively.

Sequence and phylogenetic analysis of BdPho
The cDNA fragment of BdPho (GenBank accession no. KY038166) was amplified by PCR (Fig. 1a). The 365 bp cDNA sequence has an ORF of 282 nucleotides encoding a protein of 93 amino acid residues. The predicted molecular mass and isoelectric point of BdPho peptide were 9.83 kDa and 7.54, respectively. The predicted tertiary structure showed that BdPho contained a conserved "αββ" structure ( Fig. 1b) that is widely distributed in insect defensin family. The first 22 amino acids at the N-terminus of BdPho were predicted as a signal peptide (Fig. 2), suggesting that phormicin in B. dorsalis is a secretory peptide. Two α-helix regions and three cysteine disulfide bonds were found at the mature peptide region of BdPho (Fig. 2).
The phylogenetic analysis showed that phormicins in dipteran, hemipteran, and hymenopteran species were separated into three corresponding clades (Fig. 3). The BdPho was closely related to the phormicin of olive fruit fly (B. oleae) with a bootstrap value of 96%.

Spatiotemporal expression profiles of BdPho
The results of developmental expression profiles of BdPho showed that the highest and lowest mRNA levels  AIL24687). The signal peptide of BdPho is on a gray background. The conserved mature peptide cleavage sites "-KR-" are boxed, and amino acids corresponding to predicted α-helix are underlined. Identical residues are indicated by * under the residues. The triangles indicate the six conserved cysteine residues and the dashed lines indicate the disulfide bonds occurred in adult and egg, respectively (Fig. 4a), besides, the mRNA level in adult stage was significantly higher than that in egg stage. As for adult body parts, the highest mRNA level of BdPho was found in the abdomen in both females and males, but the significant difference was only found between abdomen and head in females. The relative expression of BdPho in thorax and abdomen of female adult was significantly higher than that in male adult (Fig. 4b). For different tissues, the highest mRNA level of BdPho was observed in the fat body compared with those in Malpighian tubule and midgut, regardless of the sex (Fig. 4c).

Transcriptional response of BdPho to immune challenge and mating
As shown in Fig. 5a, the relative mRNA levels of BdPho significantly increased up to 7.46-and 14.53-fold at 3, and 6 h after PGN-EB injection compared to water injection, respectively. The expression of BdPho was also up-regulated at 9 h post PGN-EB injection, but without significant differences. The injection of PGN-SA did not show significantly induction of BdPho at 3, 6 and 9 h post injection (Fig. 5a). The relative mRNA level of BdPho significantly increased up to 3.83-fold at 12 h after starting mating compared to that of virgin females (Fig. 5b). . Data are presented as mean ± SE (n = 4). Lowercase letters above black bars and uppercase letters above white bars indicate statistical difference by ANOVA followed by the Tukey's multiple comparison test (P < 0.05), respectively. Significant differences between female and male determined with t-test, asterisks indicate significant differences in relative expression (*P < 0.05; **P < 0.01), and NS means no significant differences

Discussion
In the present study, we identified the cDNA sequence of a gene encoding phormicin in B. dorsalis. This phormicin belongs to the insects defensin family which are small cationic peptides and full of cysteines. Usually, defensins contain three peptide regions: signal peptide, prodefensin, and the mature peptide. As previously reported, the cleavage recognition sites between prodefensin and mature peptide were '-KR-' (Lowenberger et al. 1999). BdPho peptide also contained this conserved '-KR-' cleavage site. Multiple sequence alignments showed that BdPho has a high degree of identity (87.5-97.5%) with other insect phormicins and defensins in mature peptides, particularly the six conserved cysteine residues, which form three disulfide bridges. These three disulfide bonds adopted a 'cysteine stabilized αβ' (CS αβ) scaffold (Cornet et al. 1995), and the predicted tertiary structure also showed this conserved "αβ" structure in BdPho (Fig. 1b). The predicted pairing patterns of these six conserved cysteine residues were the same as that of BdDef (C1-C4, C2-C5 and C3-C6) (Liu et al. 2017), and such strong conservation suggested that these cysteines and tertiary structures might have an important structural function in these proteins.
Previous studies demonstrated that the net positive charges carried by AMPs were associated with antimicrobial activities against diverse microbes (Wang et al. 2013b). Although different AMPs have different mechanisms and modes of action, the electrostatic attraction between the cationic AMPs and negatively charged microbial membranes was the first step of the interaction between AMPs and microbes (Yeung et al. 2011). In this study, we found that BdPho mature peptide possesses + 2.9 charges at pH 7.0 condition. Depending on these surplus positive charges, BdPho may easily react with negatively charged microbial membranes by electrostatic attraction.
Different insects can produce different AMPs, and the transcription levels of particular AMPs differ a lot among species of insect. Between different developmental stages, tagmata, and tissues of a specific insect, expression profiles of AMPs are variable. For instance, BmDefA is highly expressed in early pupa and adult stages of Bombyx mori ). Similarly, a high level of constitutive expression of DmDef was found in pupa and adult stages of D. melanogaster (Dimarcq et al. 1994). However, in our previous study, BdDef was highly expressed in larva and adult stages of B. dorsalis (Liu et al. 2017). In contrast to BdDef, BdPho was highly expressed in adult stage, but lowly expressed in egg, larva and pupa stages. Although BdDef and BdPho belong to the same defensin family, their different expression profiles probably suggest a different functional involvement in the immune system during the development of B. dorsalis. The results also indicated that BdPho might play important roles in fighting against invading microbes in the adult stage.
AMPs are expressed constitutively or induced by microorganisms infection (Lemaitre and Hoffmann 2007). Most insect AMP genes are mainly expressed in immune-related organs such as the fat body, midgut and haemolymph (Wang et al. 2006). Meanwhile, the induction of different insect AMPs, as well as induction in different tissues including fat body, reproductive tissues, midgut, and salivary gland in various insects have been reported (Liao et al. 2015;Tzou et al. 2002;Wang et al. 2013b). Furthermore, it has been well documented that the expression profiles of AMPs in different tissues are related to the category of AMP genes with variations Relative mRNA levels of BdPho after mating stimulation. Data represent mean relative level values (mean ± SE) to reference genes of three independent biological replicates. The two-tailed, unpaired t-test was used to test significance. Asterisks indicate significant differences in relative expression (*P < 0.05; **P < 0.01) across insect species. In B. mori, BmDefA was highly expressed in the hemocyte, silk gland and ovary, and lowly expressed in the fat body, Malpighian tubule and midgut ). In contrast, BtDef was highly expressed in midgut and salivary, and expressed at a relatively lower level in the ovary and fat body in Bemisia tabaci (Wang et al. 2013b). In the current study, BdPho was expressed at a significantly high level in fat body, and at a much lower level in midgut, Malpighian tubule, testis and ovary. These results suggest that BdPho undertake its roles mainly in the fat body of adults to protect B. dorsalis from pathogens infection.
Though some AMPs failed to be induced in Drosophila (Hanson et al. 2016), injection of PGN has been considered as an effective way to investigate insect immune response (Neyen et al. 2014). The time course of transcription of BdPho was determined at 3-9 h after injection with two kinds of PGNs in female adult of B. dorsalis. The transcription level of BdPho was significantly induced at 3 h after PGN-EB challenge and reached a higher level at 6 h post injection; while injection of PGN-SA could not significantly stimulate the expression of BdPho from 3 to 9 h post injection. These findings implied that BdPho could be sharply induced by Gram-negative PGN but weakly induced by Grampositive PGN. The pattern of induction of BdPho is similar with that of BdDef in B. dorsalis (Liu et al. 2017) and Mdde in M. domestica (Wang et al. 2006). The BdDef was also strongly induced by PGN-EB at 3 and 6 h, but the difference was that BdDef could be significantly induced by PGN-EB at 9 h and PGN-SA at 6 h. Though BdPho and BdDef belong to the same family and have similar expression profiles in tagmata and tissues of B. dorsalis, the different induction patterns suggested that BdPho and BdDef possess different response intensity under the immune challenge of invaded bacteria. Similarly, the transcription of the Mdde was increased at 3 h post injection of bacteria, and the peak appeared at 36 h post challenge with Gram-negative bacterium (E. coli), while at the same time, injection of S. aureus (Gram-positive) did not induce expression of Mdde from 3 to 48 h. On the other hand, some differences occurred between the induction of BdPho by PGN-EB and the induction of Mdde by E. coli. For instance, infection of E. coli could strongly induce Mdde from 3 to 48 h, and the peak appeared at 36-48 h post infection, whereas injection of PGN-EB could only strongly induced BdPho at 3 and 6 h post challenge, and the peak appeared at 6 h post injection. The differences might be due to injection of massive pure PGN-EB could induce BdPho quickly while injection of E. coli required the expansion of bacteria to induce BdPho. It might also be due to different responses of innate immune system to E. coli (containing PGN and some other components) and PGN-EB.
Mating is considered as an indispensable physiological process in insects, and immunity is the basic aspect for individual survival. Thus, both immunity and mating are closely linked to fitness, and several studies reported the interactions between these two processes (Lawniczak et al. 2007). As previously reported, mating can have two different effect on immunity: either suppress or increase immunity. It was reported that there were tradeoffs between reproduction and immunity, and an increase in reproduction is generally coupled with a decrease in immunity (Fedorka et al. 2007). Mating leading to a reduction of immunity has been reported in Tenebrio molitor (Rolff and Siva-Jothy 2002), Atta colombica (Baer et al. 2006) and Matrona basilaris japonica (Siva-Jothy et al. 1998). However, some evidence suggested that immunity of insect might also increase after mating. For example, mating resulting in enhancement of immunity has been reported in Gryllus texensis (Shoemaker et al. 2006) and D. melanogaster (Domanitskaya et al. 2007;Peng et al. 2005). In this study, the results found that mating enhanced the immunity in B. dorsalis by upregulation of the AMP gene such as BdPho, which might protect the host from immune challenge during mating. This up-regulation of AMP gene may result from transfer of sperm and male accessory protein such as sex-peptide to the female during mating (Lawniczak and Begun 2004;Peng et al. 2005).
Taken all together, we cloned and characterized a phormicin gene from B. dorsalis, and investigated the spatiotemporal expression patterns of BdPho. Furthermore, we determined the mRNA levels of BdPho in female adults in response to peptidoglycan challenge and mating stimulation, which lays the foundation for understanding the molecular mechanisms in immune system and may help us to make use of insect pathogens for pest control. Furthermore, the basic knowledge derived from this study may provide information for extension the source of novel antibiotics.