Introduction
Lacking an adaptive immune system like that of vertebrates, insects rely exclusively on their effective innate immune response system to eliminate invading pathogens (Eleftherianos & Revenis, Reference Eleftherianos and Revenis2011). Fat body as well as hemolymph are important tissues in insect that are involved in defense responses against pathogens (Bao et al., Reference Bao, Lv, Liu, Xue, Xu and Zhang2010). Most of hemolymph proteins are synthesized in fat body, like antimicrobial peptides (AMP) and other humoral response molecules essential for insects’ immunity (Ferrandon et al., Reference Ferrandon, Imler, Hetru and Hoffmann2007; Arrese & Soulages, Reference Arrese and Soulages2010; Bao et al., Reference Bao, Lv, Liu, Xue, Xu and Zhang2010; Tian et al., Reference Tian, Guo, Diao, Zhou, Peng, Cao, Ling and Li2010).
Melanization, involving the rapid synthesis and deposition of melanin at a site of infection or injury, is an important component of the innate immune response in insects (Cerenius et al., Reference Cerenius, Lee and Söderhäll2008; Eleftherianos & Revenis, Reference Eleftherianos and Revenis2011). The melanin can physically shield an intruder and therefore efficiently restrain its growth, and it seems more importantly that highly reactive and toxic quinone substances and other short-lived reaction intermediates are also produced during melanin formation that kill intruding pathogens (Cerenius & Söderhäll, Reference Cerenius and Söderhäll2004; Nappi & Christensen, Reference Nappi and Christensen2005; Jiravanichpaisal et al., Reference Jiravanichpaisal, Lee and Söderhäll2006; Cerenius et al., Reference Cerenius, Lee and Söderhäll2008). Phenoloxidase (PO) is a key enzyme in melanin biosynthesis, which catalyzes the oxidation of monophenols and o-diphenols to quinones, leading to the formation of melanin (Söderhäll & Cerenius, Reference Söderhäll and Cerenius1998; Nappi & Ottaviani, Reference Nappi and Ottaviani2000; Sugumaran, Reference Sugumaran2002; Cerenius & Söderhäll, Reference Cerenius and Söderhäll2004; Christensen et al., Reference Christensen, Li, Chen and Nappi2005; Eleftherianos & Revenis, Reference Eleftherianos and Revenis2011).
Generally, PO is present as a zymogen, prophenoloxidase (PPO). The PPO cascade is activated after limited proteolysis, once it is triggered by certain microbial cell wall components, such as peptidoglycans, lipopolysaccharides, and β-1,3-glucans (Ashida & Brey, Reference Ashida, Brey, Brey and Hultmark1998; Cerenius et al., Reference Cerenius, Lee and Söderhäll2008). Previous researches have shown that, besides in hemocytes, PPO mRNA is also transcribed in fat body (Cui et al., Reference Cui, Luckhart and Rosenberg2000; Shelby & Popham, Reference Shelby and Popham2008; Feng et al., Reference Feng, Huang, Song, Stanley, Lü, Zhang and Huang2011). In arthropods, the number of PPO genes varies among different species and several PPO genes have been cloned and used to further investigate gene function (Cerenius et al., Reference Cerenius, Bangyeekhun, Keyser, Söderhäll and Söderhäll2003; Tang et al., Reference Tang, Kambris, Lemaitre and Hashimoto2006; Liu et al., Reference Liu, Jiravanichpaisal, Cerenius, Lee, Söderhäll and Söderhäll2007; Amparyup et al., Reference Amparyup, Charoensapsri and Tassanakajon2009). In most crustaceans, only one PPO gene has been reported, while in insects, there are multiple PPO subunits (Jiang et al., Reference Jiang, Wang, Korochkina, Benes and Kanost1997; Asano & Ashida, Reference Asano and Ashida2001; Sezaki et al., Reference Sezaki, Kawamoto and Asada2001; Taft et al., Reference Taft, Chen, Li and Christensen2001; Christophides et al., Reference Christophides, Vlachou and Kafatos2004; Kim et al., Reference Kim, Yao, Han, Christensen and Li2005; Doucet et al., Reference Doucet, Béliveau, Dowling, Simard, Feng, Krell and Cusson2008; Li et al., Reference Li, Wang, Jiang and Deng2009). However, there is even little attention devoted to PPO from orthopteran, let alone the PPO expression patterns from fat body of orthopteran.
Catantops pinguis (Stål) are worldwide pests in agriculture. They have spread in temperate zones and subtropical zones, and are common in Africa and south and east Asia. They bring about considerable damage to crops, such as Gramineae, Leguminosae, and Compositae, because of their complex feeding habits. In the present study, two PPO cDNA sequences from fat body of C. pinguis were cloned and sequenced. The two PPOs were characterized in terms of their tissue distribution and expression patterns in response to infection by Escherichia coli strain DH 5α. Furthermore, PPO mRNA transcripts from fat body in different developmental stages and different months were detected. These results suggest that PPO is essential for locust survival in response to challenge by E. coli and for its growth.
Materials and methods
Insect rearing and tissue isolation
C. pinguis were obtained from the tea plantation of Hangzhou in Zhejiang province, China. They were reared at 25±1 °C, under a photoperiod of 14/10 h (light/dark) and 65–70% relative humidity in an artificial climatic chamber. Locusts were kept in a group of 200 individuals in each large cage (55×55×55 cm), and fed daily with fresh wheat seedlings supplemented with bran. The larval instars were determined by molting.
About 600 eggs of C . pinguis per batch were used for hatching. Fat bodies were dissected from different developmental stages in a sterile operation. Eleven tissues including fat body were isolated from other adults for tissue analysis. Wild-caught adults at the beginning, middle, and end of each month were also used for fat bodies dissections. All tissue samples were stored at −80 °C until further use.
RNA isolation, cDNA synthesis, and polymerase chain reaction (PCR)
Total RNA was extracted from the fat body using the Trizol reagent (Invitrogen), and contaminating genomic DNA was eliminated with RNase-free DNase (Promega). The purified mRNA was used to synthesize first-strand cDNA using AMV reverse transcriptase (Takara) according to the manufacturer's protocol. First-strand cDNA was generated in a 25-μl reaction volume containing 1 μg total RNA, 2 pM oligo(dT18), 20 U RNase inhibitor, and 5 U AMV reverse transcriptase.
Four degenerate primers (table 1) for PCR were designed based on the highly conserved sequences of the conserved regions of entomic PPO1 and PPO2 gene sequences, respectively, from Tribolium castaneum (NM_001039404, NM_001039433), Tenebrio molitor (AB020738), Anopheles gambiae (XM_312089, XM_316323), Aedes aegypti (XM_001648918, AF292113), Drosophila melanogaster (AB055857), Bombyx mori (NM_001043870, NM_001044069), Apis mellifera (NM_001011627), Holotrichia diomphalia (AB079665), and Manduca sexta (O44249) (http://www.ncbi. nlm.nih.gov) using the Primer 5 software. PCR amplification of the target cDNA fragments was performed in reaction mixtures containing 1 μl of the fat body cDNA, 10 pmol of forward and reverse primers, 1 U of Platinum Taq High Fidelity DNA polymerase, 10×high-fidelity PCR buffer, and sterile H2O to a final volume of 25 μl. The PCRs were performed as follows: an initial denaturation step at 94 °C for 5 min, followed by three cycles of denaturation at 94 °C for 30 s, annealing at 45 °C for 30 s, and elongation at 72 °C for 90 s, and then 28 cycles of denaturation at 94 °C for 30 s, annealing at 48 °C for 30 s, and elongation at 72 °C for 90 s, followed by a 10 min extension at 72 °C and cooling to 12 °C. PCR products were analyzed by 1.0% agarose gel electrophoresis and each fragment corresponding to the expected size of approximately 1100 bp was purified and subcloned into the pMD18-T simple vector (Takara). After being transformed into E. coli DH 5α competent cells, positive clones were sequenced, and partial cDNA sequences of CpPPO1 and CpPPO2 were obtained using the pair of degenerate primers.
Table 1. Primer sequences used for amplification of CpPPO1 and CpPPO2.
1 F: forward, R: reverse.
2 D: degenerate, G: gene-specific primer.
3 B: T/C/G; K: G/T; S: C/G; Y: C/T; M: A/C; V: A/C/G; N: A/C/T/G; R: A/G; W: A/T; D: A/T/G.
Rapid amplification of cDNA ends (RACE)
To obtain 5′- and 3′-end sequences, 1 μg of purified total RNA from the fat body was used for the preparation of 3′-RACE-Ready and 5′-RACE-Ready cDNA templates using the BD SMART RACE cDNA Amplification Kit (Clontech, USA) according to the manufacturer's protocol. Two internal gene-specific primers for each 3′-RACE and 5′-RACE PCR (table 1) were designed according to the gene segments obtained. PCR mixtures were used and the primary PCR amplification profiles were similar to the program described before, while the annealing temperature was set at 55 °C. After agarose gel electrophoresis, the RACE products were purified and cloned into the pMD18-T vector (Takara). Positive colonies were selected and sequenced completely.
Sequences analysis and phylogenetic tree construction
Similarity searches of cDNA and deduced amino acid sequences were performed using the BLASTN and BLASTP programs (http://www.ncbi.m.nih.gov/blast). Protein sequences analyses (such as putative signal peptide and N-glycosylation sites) were performed with various online software, such as SignalP3.0 Server and NetNGlyc (http://www.cbs.dtu.dk/services/). A multiple-sequence alignment was performed using the ClustalW program (Vector NTI 11.0, Invitrogen) and phylogenetic analysis was performed using maximum likelihood method analysis with the Mega 5 software.
Tissue distribution analysis
Total RNA was isolated from the fat body, epidermis, muscle, Malpighian tubule, heart, ovary, testis, trachea, midgut, hindgut, and brain. After eliminating contaminated DNA, total RNA was used for cDNA synthesis.
Expression patterns of CpPPO1 and CpPPO2 were analyzed by semi-quantitative using reverse transcription–PCR (RT–PCR) combined with oligonucleotide probe hybridization. Gene-specific primers for CpPPO1 and CpPPO2 (CpPPO1-PF, CpPPO1-PR, CpPPO2-PF, and CpPPO2-PR; table 1) were designed. β-Actin was used as an internal control. PCRs were performed as described above. The PCR products were subsequently blotted onto Hybond-N+ membranes (Amersham, UK) with downward capillary action in 20×SSC transfer buffer (3 M NaCl, 0.3 M NaC6H5O7·2H2O, pH 7.0) overnight. Suitable cDNA fragments (CpPPO1, 700 bp; CpPPO2, 614 bp) (fig. 1a, b) were labeled with DIG-High Prime and used as probes. Membranes were pre-hybridized at 42 °C for 0.5 h and incubated in the DIG-high prime-labeled probes for 6 h in hybridization buffer containing 50% formamide, 5×Denhardt's solution, and 0.1% SDS. After hybridization, the membranes were washed in 2×SSC and 0.5×SSC twice. Then, the hybridized probes were immunodetected with anti-digoxigenin-AP Fab fragments and visualized with the colorimetric substrates NBT/BCIP.
Fig. 1. Nucleotide and deduced amino acid sequences of CpPPO. (a) Nucleotide and deduced amino acid sequences of CpPPO1. (b) Nucleotide and deduced amino acid sequences of CpPPO2. Translation initiation and termination sites are indicated in bold and italic font. The putative conserved copper-binding regions are underlined and the conserved histidine residues are boxed. The completely conserved thiol ester motif region (GCGWPQHM) is indicated in grey shading. The N-glycosylation sites are indicated with a dotted curve. Polyadenylation signal sites are indicated. Two allele-specific primers are marked by arrowheads. An arrow indicates a possible cleavage site for signal peptidase and the signal peptide is double underlined.
Bacteria challenge and expression profiles of CpPPO1 and CpPPO2
The insect pathogenic bacterium E. coli strain DH 5α was cultured on an Amp− LB plate and incubated at 37 °C overnight. A single colony was picked and placed in LB media and incubated overnight in a shaking incubator at 37 °C and 254 rpm. Bacterial cultures were then centrifuged (5000 rpm, 4 °C) and the pellets were resuspended in 0.2 M PBS buffer Phosphate-Buffered Saline. Bacterial density in the PBS buffer was adjusted to obtain an optical density (OD) of 0.6. Five-day-old adults were chosen for immune challenging analysis by E. coli. Each was injected with 2 μl bacterial suspension. Ten locusts from each treatment were sampled at 0, 6, 12, 24, 36, and 48 h after the immune challenge began. Total RNA from the fat body dissected from the samples was isolated and cDNA was synthesized. A semi-quantitative determination of CpPPO1 and CpPPO2 was carried out as described above.
Temporal expression analysis of CpPPO1 and CpPPO2 transcripts
Total RNA extracted from fat bodies in different stages, including the first, second, third, fourth, and fifth instar larvae and adults, and fat bodies from adults caught in different months were prepared for cDNA synthesis. Temporal expression levels of CpPPO1 and CpPPO2 transcripts were assessed by semi-quantitative detection, as described above.
Results
Characterization of PPO1 and PPO2 from C. pinguis
Two distinct homologous PPO genes of C. pinguis were cloned using RT–PCR and RACE. The full-length cDNA of CpPPO1 is 2347 bp, including an open reading frame (ORF) of 2187 bp encoding a predicted protein of 728 amino acid residues with a predicted molecular mass of approximately 84.26 kDa and a theoretical pI of 6.91. Translation initiation occurred at an ATG codon, while termination occurred at a TGA codon. A single polyadenylation signal was located in the 3′-UTR, beginning at 2358 bp. Possible N-glycosylation sites were predicted at positions 25 and 595 (fig. 1a). The complete sequence of CpPPO2 was 2445 bp, including an ORF of 2076 bp encoding a predicted protein of 691 amino acid residues with a predicted molecular mass of approximately 79.66 kDa and a theoretical pI of 6.27. Translation initiation occurred at an ATG codon, while termination occurred at a TAA codon. A single polyadenylation signal was located in the 3′-UTR, beginning at 2453 bp. Possible N-glycosylation sites were predicted at positions 83, 293, 463, 543, and 660 (fig. 1b). According to the Signal P3.0 Server on line, the N-terminus of deduced PPO protein assumes the characters as a pupative signal peptide including 21 amino acid residues (fig. 1a), but no sufficient data to support the result at present.
Multiple sequence alignment and phylogenetic analysis
Alignment of PPO proteins revealed that both CpPPO protein sequences contained three highly conserved features: two highly conserved copper-binding regions, six histidine residues, and a thiol-ester motif (fig. 2). For CpPPO1, six conserved histidine residues, which are likely to bind two copper ions, were present at conserved sites within the two copper-binding sites: copper A (H243, H247, and H272) and copper B (H399, H403, and H439) (fig. 1a). For CpPPO2, six conserved histidine residues were present at the two copper-binding regions: copper A (H212, H216, and H241) and copper B (H369, H373, and H409) (fig. 1b). The highly conserved thiol ester motif (GCGWPQHM) was present in both CpPPO1 and CpPPO2 at Gly612 to Met619 and Gly584 to Met591 (fig. 1a, b), respectively. A BLASTX analysis showed that the two proteins exhibited high amino acid sequence identity (50–89%, 50–93%, respectively) with those of other insect species (tables 2 and 3).
Fig. 2. Multiple amino acid sequence alignment of CpPPOs with those of other insects. Alignments of CatpiPPO1, CatpiPPO2 (FJ598047), AnogaPPO1 (XM_312089), SarbuPPO1 (AF161260), TricaPPO1 (NM_001039404), BommoPPO1 (NM_001043870), ChofuPPO1 (EU046570), PieraPPO1 (HM776513), AnogaPPO2 (XM_316323), SarbuPPO2 (AF161261), BommoPPO2 (NM_001044069), TricaPPO2 (NM_001039433), ChofuPPO2 (EU046573), CamflPPO (GL435066), ApimePPOA3 (NM_001011627) and PieraPPO2 (HM235814) proteins were performed using the Vector NTI 11.0 software.
Table 2. The related information of PPO gene family and the identity (%) to CpPPO1.
Table 3. The related information of PPO gene family and the identity (%) to CpPPO2.
The evolutionary relationship among CpPPO1 and CpPPO2 amino acid sequences with other arthropod PPOs was assessed by the maximum likelihood method, using Mega 5 (fig. 3). There are 73 PPO sequences in the tree that form two different groups including the crustacean PPOs and the insect PPOs. The insect PPOs group can also be separated into five distinct clades: diptera PPO, coleopteran PPO, Lepidoptera PPO, hymenoptera PPO, and orthoptera PPO. According to the dendogram, CpPPO1 and CpPPO2 branched within the clade of PPOs of orthoptera, and separately from other insect orders. Within orthoptera, only two PPO gene sequences were obtained for each insect species. CpPPO1 and CpPPO2 showed exceptionally high homology with LmPPO1 and LmPPO2, respectively. The phylogenetic analysis also showed that PPOs from hemiptera were more closely related to PPOs from orthoptera than from coleoptera (fig. 3).
Fig. 3. Phylogenetic relationships of CpPPO1 and CpPPO2 with PPOs from other insects. The phylogenetic tree was constructed on the basis of known amino acid sequences using a maximum likelihood method analysis and the Mega 5 software. A bootstrap analysis was carried out and the robustness of each cluster was verified using 100 replicates. Values at the cluster branches indicate the results of the bootstrap analysis. Insect PPOs: Locusta migratoria (LocmiPPO1: FJ771025; LocmiPPO2: FJ771024); Catantops pinguis (CatpiPPO1; CatpiPPO2: FJ598047); Bombyx mori (BommoPPO1: NM_001043870; BommoPPO2: NM_001044069); Bombyx mandarina (BommaPPO1: EU569724; BommaPPO2: EU047703); M. sexta (MansePPO: O44249); H. cunea (HypcuPPO: HCU86875; HypcuPPO2: AF020391); Heliothis virescens (HelviPPO1: DQ841706; HelviPPO2: EF044308); Spodoptera frugiperda (SpofrPPO1: DQ289581; SpofrPPO2: DQ289582); Pieris rapae (PieraPPO1: HM776513; PieraPPO2: HM235814); Plutella xylostella (PluxyPPO1: GU214206; PluxyPPO2: GU214207); Choristoneura fumiferana (ChofuPPO1: EU046570; ChofuPPO2: EU046573); Galleria mellonella (GalmePPO: AF336289; GalmePPO2: AY371489); Plodia interpunctella (PloinPPO: AY665397); Ostrinia furnacalis (OstfuPPO: DQ333883); Helicoverpa armigera (HelarPPO2: DQ114946); Spodoptera exigua (SpoexPPO2: EF684939); Spodoptera litura (SpoliPPO: AY703825); Biston betularia (BisbePPO2: GU953227); Culex quinquefasciatus (CulquPPO: XM_001848913; CulquPPO1: XM_001846507); A. aegypti (AedaePPO: XM_001648918; AedaePPO2: AF292113; AedaePPO3: AF310673); D. melanogaster (DromePPOA3: AB055857); A. gambiae (AnogaPPO1: XM_312089; AnogaPPO2: XM_316323; AnogaPPO3: XM_315073; AnogaPPO4: XM_315084); Musca domestica (MusdoPPO: AY494738); Armigeres subalbatus (ArmsuPPO1: AF260567; ArmsuPPO3: AY487171; ArmsuPPO4: AY487172; ArmsuPPO5: DQ862064; ArmsuPPO6: DQ862065); Anopheles stephensi (AnostPPO: AF062034); Sarcophaga bullata (SarbuPPO1: AF161260; SarbuPPO2: AF161261); T. castaneum (TricaPPO1: NM_001039404; TricaPPO2: NM_001039433); T. molitor (TenmoPPO: AB020738); H. diomphalia (HoldiPPO1: AB079664; HoldiPPO2: AB079665); Harpegnathos saltator (HarsaPPO: GL446009); Camponotus floridanus (CamflPPO: GL435066); A. mellifera (ApimePPOA3: NM_001011627). Crustacean PPOs: Daphnia magna (DapmaPPO: FJ381649); Oratosquilla oratoria (OraorPPO: HQ588346); Litopenaeus vannamei (LitvaPPO: EF115296; LitvaPPO1: EU284136; LitvaPPO2: EF565469); Marsupenaeus japonicus (MarjaPPO: AB073223); Cherax quadricarinatus (ChequPPO: JQ040507); Penaeus monodon (PenmoPPO: AF521948; PenmoPPO2: EU853256); Eriocheir sinensis (ErisiPPO: EF493829); Portunus trituberculatus (PortrPPO: FJ215871); Homarus gammarus (HomgaPPO: AJ581662); Homarus americanus (HomamPPO: AY655139); Fenneropenaeus chinensis (FenchPPO: FJ594415); Macrobrachium rosenbergii (MacroPPO: DQ182596); Scylla serrata (ScysePPO: DQ435606); Panulirus longipes (PanloPPO: GQ240941).
Expression analysis of CpPPO1 and CpPPO2 in different tissues
Total RNA was extracted from the fat body, epidermis, muscle, Malpighian tubule, heart, ovary, testis, trachea, mid-gut, hindgut, and brain of adults. The expression patterns of CpPPO1 and CpPPO2 were assessed by semi-quantitative analysis (fig. 4a). The CpPPO1 mRNA transcript was expressed in all the examined tissues except the epidermis, muscle, ovary, and brain, and the highest expression level was detected in the fat body. The CpPPO2 mRNA was transcribed in all the examined tissues except the ovary and a high expression level was detected in the fat body, while a very low level was observed in the mid-gut. In comparison, a higher expression level of CpPPO2 was observed in the same tissue.
Fig. 4. Expression patterns analysis of CpPPO1 and CpPPO2 by semi-quantitative RT–PCR combined with oligonucleotide probe hybridization. (a) Tissue distribution analysis of CpPPO1 and CpPPO2: fat body (Fb), epidermis (Ep), muscle (Mu), Malpighian tubule (Mt), heart (H), ovary (O), testis (Te), trachea (Tr), midgut (Mg), hindgut (Hg), and brain (Br). β-Actin was used as a control housekeeping gene to indicate and standardize the amount of cDNA template in each RT–PCR. Specific probes were labeled with digoxin and used for hybridization and detection of target gene fragments. (b) Effect of bacterial challenge on expression of CpPPO1 and CpPPO2 transcripts. Fat bodies were collected from adult C. pinguis at 0, 6, 12, 24, 36, and 48 h after infection by E. coli. (c) CpPPO1 and CpPPO2 transcripts in different developmental stages. Fat bodies were collected from 1st instar larvae to adults of C. pinguis. (d) CpPPO1 and CpPPO2 mRNA expression patterns in C. pinguis fat bodies collected from different months. Fat bodies were collected from adults from January to December.
Bacterial challenge and expression profiles of CpPPO1 and CpPPO2
To understand the influence of E. coli on locust immunity, expression levels of CpPPO1 and CpPPO2 mRNA were determined from 0 (untreated, CK), 6, 12, 24, 36, and 48 h after E. coli injection in adults (fig. 4b). The expression level of CpPPO1 was low at 0, 6, and 12 h after E. coli injection, then significantly up-regulated at 24 h, and total two peaks of CpPPO1 transcripts at 24 and 48 h after injection were observed. The transcript level of CpPPO2 mRNA was significantly increased at 24 and 48 h after E. coli injection, while a slight decrease was seen at 36 h after treatment.
Temporal expression analysis of CpPPO1 and CpPPO2 transcripts
The developmental expression profiles of CpPPO1 and CpPPO2 were investigated. Both CpPPO1 and CpPPO2 mRNAs were constitutively transcribed during different developmental stages, from the first instar nymphae (fig. 4c). High transcript levels of CpPPO1 mRNA were measured in the first instar and the earlier stages of second instar larvae, while the transcript was undetectable at day 6 of second instar larvae, and days 2 and 5 of fourth instar larvae. Then, the CpPPO1 mRNA transcript level increased significantly at day 8 of fourth instar larvae and stayed high in fifth instar larvae before their molt. The CpPPO1 mRNA transcript level was significantly decreased in day 1 adults and remained at a low level. The CpPPO2 mRNA was persistently transcribed during all the developmental stages examined. High expression levels of CpPPO2 were detected in the earlier stages of first, second, third, and fourth instar larvae, the later stages of fifth instar larvae and adults. However, the transcript level of CpPPO2 mRNA was extremely low at day 8 of fourth instar larvae.
As C. pinguis are overwintering insects, we also investigated the expression profiles of CpPPO1 and CpPPO2 in different months of the year (fig. 4d). A high transcript level of CpPPO1 was detected in a couple of months from January to July along with December, relatively, except in early April. Expression of CpPPO1 mRNA decreased from August and remained low until later November, and an extremely low level was detected in early November. CpPPO2 mRNA was abundantly expressed during the whole year, with extremely high levels in January, early May, late July, late October, and December.
Discussion
The fat body is the most prominent organ in insects. It plays major roles in the intermediary metabolism, in the energy storage, in the innate immunity and is responsible for the synthesis and supply of hemolymph compounds (Antonova et al., Reference Antonova, Alvarez, Kim, Kokozak and Raikhel2009; Cardoso et al., Reference Cardoso, Cres, Moura, de Almeida and Bijovsky2010; Martins et al., Reference Martins, Serrão, Ramalho-Ortigão and Paolucci-Pimenta2011; Roy & Raikhel, Reference Roy and Raikhel2011). The fat body can regulate the concentration in the hemolymph substances and participate in insect homeostasis (Haunerland & Shirk, Reference Haunerland and Shirk1995; Arrese & Soulages, Reference Arrese and Soulages2010; Martins et al., Reference Martins, Serrão, Ramalho-Ortigão and Paolucci-Pimenta2011).
In the present research, we report two homologous PPO genes from fat body of C . pinguis (Stål), which were designated CpPPO1 and CpPPO2. Like other insect PPO genes, both CpPPO1 and CpPPO2 contain two putative tyrosinase copper-binding motifs with six highly conserved histidine residues and a highly conserved thiol ester-like motif (GCGWPQHM), whose function has not been reported. Although it was predicted that there is a pupative signal peptide, consisting of 21 amino acids, in CpPPO1 by signal P3.0 server, there was no further work to support the result at present, in addition, no hydrophobic signal peptide has been reported in other arthropods to date (Aspan et al., Reference Aspan, Huang, Cerenius and Söderhäll1995; Fujimoto et al., Reference Fujimoto, Okino, Kawabata, Iwanaga and Ohnishi1995; Cho et al., Reference Cho, Liu, Lee, Kuo, Chang, Liu and Chen1998; Lee et al., Reference Lee, Ahmed, della Torra, Kobayashi, Ashida and Brey1998; Cui et al., Reference Cui, Luckhart and Rosenberg2000; Ling & Yu, Reference Ling and Yu2005; Shelby & Popham, Reference Shelby and Popham2008; Tsao et al., Reference Tsao, Lin, Christensen and Chen2009; Feng et al., Reference Feng, Huang, Song, Stanley, Lü, Zhang and Huang2011). Probably, further researches should be carried out concerning the structure and function of PPO1 protein.
Multiple alignments of the deduced proteins of the two genes CpPPO1 and CpPPO2 showed a high relevant identity of 89% and 93% with LmPPO1 (FJ771025) (table 2) and LmPPO2 (FJ771024) (table 3), but they shared less identity, 57%, with each other. Previous studies have revealed that the PPOs from one species are less homologous to each other than to PPOs from other species, suggesting that duplication and divergence of the PPO genes occurred prior to the emergence of these distinct insect species (Cui et al., Reference Cui, Luckhart and Rosenberg2000). Similarly, PPO1 and the ancestor of other PPOs in A. gambiae may have duplicated and diverged before the separation of Diptera and Lepidoptera (Müller et al., Reference Müller, Dimopoulos, Blass and Kafatos1999).
Previous studies have shown that, besides in hemolymph, PPOs are also transcribed in insect fat body and other tissues. Our study showed that both CpPPO1 and CpPPO2 transcripts are also abundantly expressed in the fat body. CpPPO1 and CpPPO2 mRNA were also transcribed in other tissues, including the Malpighian tubule, heart, testis, trachea, midgut, and hindgut. A knockdown experiment with the Drosophila melanization proteases (MP1 and MP2) showed that melanization is crucial for the innate immune response against bacterial and fungal infections (Tang et al., Reference Tang, Kambris, Lemaitre and Hashimoto2006). In Pacifastacus leniusculus, RNA interference-mediated depletion of PPO leads to increased bacterial growth, lower phagocytosis, lower PO activity, lower nodule formation, and higher mortality when infected with a highly pathogenic bacterium, Aeromonas hydrophila. However, RNA interference of the aninhibitor of PPO results in lower bacterial growth, increased phagocytosis, increased nodule formation, higher PO activity, and delayed mortality (Liu et al., Reference Liu, Jiravanichpaisal, Cerenius, Lee, Söderhäll and Söderhäll2007). These studies further demonstrate that the PPO-activating system plays an essential role in invertebrate defense against diverse pathogens (Cerenius & Söderhäll, Reference Cerenius and Söderhäll2004). In our study, CpPPO1 and CpPPO2 transcript levels are transiently and significantly influenced after E. coli infection. However, apparently contradictory results have also been reported: studies on Drosophila and A. gambiae revealed that PO activity was redundant in response to several bacterial and fungal pathogens (Leclerc et al., Reference Leclerc, Pelte, EI Chamy, Martinelli, Ligoxygakis, Hoffmann and Reichhart2006; Schnitger et al., Reference Schnitger, Kafatos and Osta2007).
Insect fat body has been demonstrated to synthesize various AMPs and PPO mRNA transcript was also detected in it. However, there is little report involving PPO expression pattern in fat body.
For many insect species, there are several distinct PPO genes, and each may have different physiological functions or cooperative interactions (Jiang et al., Reference Jiang, Wang, Korochkina, Benes and Kanost1997; Müller et al., Reference Müller, Dimopoulos, Blass and Kafatos1999; Cerenius & Söderhäll, Reference Cerenius and Söderhäll2004). It had been indicated that PPO genes in A. gambiae are differentially expressed in different developmental stages (embryo to adult) (Müller et al., Reference Müller, Dimopoulos, Blass and Kafatos1999). C. pinguis undergoes several ecdyses, from eggs to the adults, and the interval duration tends to be diverse. Semi-quantitative analysis revealed that both CpPPO1 and CpPPO2 mRNAs were constitutively transcribed during different developmental stages, from first instar nymphae. High transcript levels of the two PPO genes were detected in first instar and second instar larvae, and high expression levels of PPO2 were observed in adults. In contrast, high expression levels of PPO1 and PPO2 mRNA were detected in mid-instar larvae, but not in pupae, adults, or eggs in Hyphantria cunea (Park et al., Reference Park, Shin, Kim, Park, Lee, Brey and Park1997). Intriguingly, this seems to be distinct from the situation in A. gambiae, where the PPO transcript was highly expressed in eggs, but at a low level in other developmental stages (Lee et al., Reference Lee, Ahmed, della Torra, Kobayashi, Ashida and Brey1998). In contrast, real-time RT–PCR showed higher amounts of AmPPO transcripts in adults and older pupae than in younger pupae and larvae, suggesting a function of AmPPO in adult exoskeleton melanization and differentiation (Lourença et al., Reference Lourença, Zufelato, Bitondi and Simões2005). In B. mori, there was a gender difference in the developmental changes in PPO mRNA level, because the high expression level falls in different stages between females and males (Yamamoto et al., Reference Yamamoto, Yakiyama, Fujii, Kusakabe, Koga, Aso and Ishiguro2000). This differentiation of PPO transcription during the developmental stages suggests a role of PPO in insect growth and development.
In the wild, insects often suffer when faced with poor weather conditions (Robb & Forbes, Reference Robb and Forbes2006). As wintering insects, the adults of C. pinguis were collected in the field to investigate the changing trends of their PPO mRNA transcripts and PO activity in different months. Seasonal differences between PPO1 and PPO2 mRNA transcripts were obvious: high transcript levels of CpPPO1 were detected in couple of months from January to July, along with December, except in early April and an extremely low level was detected in the early November, while the CpPPO2 mRNA was abundantly expressed in January, early May, late July, late October, and December. The variation of PPO mRNA transcripts during different months may be related to changing climate factors, such as temperature and humidity, which can dramatically influence the development and survival of many insects (Abril et al., Reference Abril, Oliveras and Gómez2010; Sgolastra et al., Reference Sgolastra, Bosch, Molowny-Horas, Maini and Kemp2010; Tamiru et al., Reference Tamiru, Getu, Jembere and Bruce2011). Moreover, food composition is another important factor playing a vital role in insect immunity (Alaux et al., Reference Alaux, Ducloz, Crauser and Le Conte2010; Fellous & Lazzaro, Reference Fellous and Lazzaro2010).
In this study, we present evidence supporting an important role for PPO in C. pinguis immunity and growth. Interestingly, according to current signal P3.0 server, the deduced CpPPO1 protein sequence contains a pupative signal peptide, which has not been reported in other insects. Clearly, further research is required to determine the structure and function of CpPPO1. Both CpPPO1 and CpPPO2 are immune-responsive genes, and their expression is tissue-specific. Distinct expression levels of CpPPO1 and CpPPO2 were detected in locusts from different developmental stages and different months. Further studies are needed to ensure the influence of certain environmental factors individually. Nevertheless, it seems clear that both CpPPO1 and CpPPO2 are important components of the C. pinguis immune defense system.
Acknowledgements
This work was supported by National Natural Science Foundation of China (Grant Nos. 30970473 and 31000880), Zhejiang Provincial Natural Science Foundation of China (Grant Nos. Y307551 and Y3100176), The Project of Zhejiang Key Scientific and Technological Innovation Team (Grant No. 2010R50039), Hangzhou Science and Technology Development Program of China (Grant No. 20081443B03 and 20110232B11), the Program for Excellent Young Teachers in Hangzhou Normal University (Grant No. JTAS 2011-01-031) and Hangzhou Normal University High-level Talents Start-up Fund (Grant No. YS05203105).
