Prophenoloxidase genes and antimicrobial host defense of the model beetle, Tribolium castaneum

Journal of Invertebrate Pathology 132 (2015) 190–200

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Prophenoloxidase genes and antimicrobial host defense of the model beetle, Tribolium castaneum Kakeru Yokoi 1, Yuuki Hayakawa, Daiki Kato, Chieka Minakuchi, Toshiharu Tanaka, Masanori Ochiai 2, Katsumi Kamiya 3, Ken Miura ⇑ Applied Entomology Laboratory, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan

a r t i c l e

i n f o

Article history: Received 24 March 2015 Revised 26 October 2015 Accepted 27 October 2015 Available online 28 October 2015 Keywords: Innate immunity Tribolium castaneum Melanin synthesis Phenoloxidase RNA interference

a b s t r a c t In this study, we characterized prophenoloxidase (proPO, (PPO)) genes of Tribolium castaneum and examined their involvement in antimicrobial host defense. Amino acid sequence comparison with wellcharacterized PPO proteins from other insect species suggested that T. castaneum PPO genes encoded functional proenzymes, with crucial sequence motifs being conserved. Developmental kinetics of the mRNA of two PPO genes, PPO1 and PPO2 in the pupal stage were different to each other. The PPO1 mRNA levels consistently decreased during pupal development while that of PPO2 peaked at mid-pupal stage. The two mRNAs also exhibited distinct responses upon immune challenges with heat-killed model microbes. The PPO1 mRNA stayed nearly unchanged by 6 h post challenge, and was somewhat elevated at 24 h. In contrast, the PPO2 mRNA significantly decreased at 3, 6 and 24 h post challenge. These trends exhibited by respective PPO genes were consistent irrespective of the microbial species used as elicitors. Finally, we investigated the involvement of T. castaneum PPO genes in antimicrobial host defense by utilizing RNA interference-mediated gene silencing. Survival assays demonstrated that double knockdown of PPO genes, which was accompanied by weakened hemolymph PO activities, significantly impaired the host defense against Bacillus subtilis. By contrast, the knockdown did not influence the induction of any of the T. castaneum antimicrobial peptide genes that were studied here, except for one belonging to the gene group that shows very weak or negligible microbial induction. PPO knockdown as well weakened host defense against Beauveria bassiana moderately but significantly depending on the combination of infection methods and targeted genes. Our results indicated that the PPO genes represented constituents of both antibacterial and antifungal host defense of T. castaneum. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction To fight against invading pathogens, insects utilize wellorganized innate immune systems, which are conveniently divided into humoral and cellular reactions (Gonzalez-Santoyo and Abbreviations: PO, phenoloxidase; PPO, prophenoloxidase; RNAi, RNA interference; qRT-PCR, real-time quantitative RT-PCR; Att, attacin; Cec, cecropin; Col, coleoptericin; Def, defensin; RPL32, ribosomal protein L32; malE, maltose binding protein E. ⇑ Corresponding author. E-mail address: [email protected] (K. Miura). 1 Present address: Insect Genome Research Unit, Agrogenomics Research Center, National Institute of Agrobiological Sciences, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan. 2 Address: Biochemical Laboratory, Institute of Low Temperature Science, Hokkaido University, Kita19, Nishi8, Kita, Sapporo 060-0819, Japan. 3 Address: Gifu Prefectural Agricultural Technology Center, Matamaru, Gifu 5011152, Japan. http://dx.doi.org/10.1016/j.jip.2015.10.008 0022-2011/Ó 2015 Elsevier Inc. All rights reserved.

Cordoba-Aguilar, 2012; Hultmark, 2003; Lavine and Strand, 2002; Lemaitre and Hoffmann, 2007). The most well-documented among insect humoral immune reactions is the massive induction of an array of antimicrobial peptide (AMP) genes in fat body in response to microbial infection (Kleino and Silverman, 2014; Valanne et al., 2011). The melanization reaction that eventually results in the deposition of insoluble melanin at the sites of infection or injury represents another hallmark of insect humoral immunity (Cerenius et al., 2008; Cerenius and Söderhäll, 2004; Nappi and Christensen, 2005). The typical cellular immune reactions, such as encapsulation or nodule formation, are frequently accompanied by melanin deposition on foreign bodies, suggesting the important roles of melanization in insect immune defense. The key enzyme for melanin synthesis, phenoloxidase (PO) exists constitutively as its zymogen form proPO (PPO) in hemolymph or certain types of hemocyte and is readily activated upon infection, and this acute nature may compensate for relatively slow

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AMP production and secretion. Upon microbial infection, pathogen-associated molecular patterns, such as peptidoglycans, b-1,3-glucans or lipopolysaccharides are sensed by specific pattern recognition receptors, which trigger the PPO-activating serine protease cascade, ultimately leading to PPO activation through the removal of its N-terminal moiety (Kanost et al., 2004; Ochiai and Ashida, 1999, 2000; Park et al., 2007; Yu and Kanost, 2004). The resultant enzymatically active PO catalyzes early steps of melanin synthesis reactions initially by hydroxylating monophenols to ortho-diphenols, which are further oxidized by this enzyme to ortho-quinones. These reactions eventually lead to the formation of insoluble melanin via paths composed of both enzymatic and non-enzymatic reactions, which physically shields intruders or helps wound healing. Importantly, cytotoxic intermediate products of melanin synthesis, such as ortho-quinones, are regarded as important effector molecules in acute phase antimicrobial immune responses (Cerenius et al., 2008; Zhao et al., 2007). Insect PPO proteins are generally synthesized in specific types of hemocytes. For example, lepidopteran hemocytes are classified into prohemocytes, granular cells, plasmatocytes, spherule cells and oenocytoids (Lavine and Strand, 2002), among which the oenocytoid is a cell type that produces PPO (Iwama and Ashida, 1986). In the case of dipteran Drosophila, the site for its production is another type of hemocyte, the crystal cell (Williams, 2007). The PPO proteins that lack a signal peptide are thought to be released into hemolymph by hemocyte eruption upon infection as well as in the absence of infection or injury with a low frequency (Cerenius and Söderhäll, 2004; Gonzalez-Santoyo and Cordoba-Aguilar, 2012). Some literature describes the relevance of observed PO activity and immune defense phenotypes in non-model insects, and the conclusions regarding the importance of melanization reaction vary from study to study (Gonzalez-Santoyo and Cordoba-Aguilar, 2012). While direct evidence for the involvement of PO in host defense are limited to insect species in which loss-of-function assays utilizing mutant lines or gene knockout/knockdown approaches are available, conclusions were not always consistent even in those cases (Binggeli et al., 2014). Collectively, respective results are likely to depend on the configurations of insect species and developmental stages, pathogen species, and targeted genes. We have been studying in recent years insect immune pathways using a model Coleoptera, Tribolium castaneum, taking advantage of its RNA interference (RNAi)-friendly nature and fullgenome information (Richards et al., 2008; Tomoyasu et al., 2008). Moreover, its potential immune-related genes have been annotated (Zou et al., 2007). By using RNAi-based gene knockdown approaches, we have so far demonstrated that T. castaneum has the functional Toll and IMD pathways that show somewhat different specificity compared to Drosophila ones and that some constituents of the IMD pathway indeed contribute to defense against some bacterial species (Koyama et al., 2015; Yokoi et al., 2012a, 2012b). In this study, we applied similar approaches to T. castaneum PPO genes. The genome of T. castaneum encodes three PPO genes, two of which, PPO2 and PPO3 share 98.8% nucleotide and 99.6% amino acid sequences in ORFs. Thus, we did not distinguish between these two genes that were collectively referred to as PPO2 in this study. Our results demonstrated different developmental profiles and responses to infection between PPO genes as well as their moderate but significant contributions in both antibacterial and antifungal immune defense. 2. Material and methods 2.1. Insect rearing T. castaneum was reared at 30 °C as described in our previous papers (Yokoi et al., 2012b). For fungal infection experiments in

191

the pupal stage, day 0 pupae were transferred to 25 °C usually just after dsRNA injection. 2.2. Microorganisms Preparation and injection of Escherichia coli, Micrococcus luteus, Saccharomyces cerevisiae, Enterobacter cloacae and Bacillus subtilis were done as described in Yokoi et al. (2012a). The former three species were used as elicitors in a form of heat-killed bacterial suspensions, and the latter two were used for survival assays as live bacteria. A strain of entomopathogenic fungus Beauveria bassiana used in this study was isolated by K.K. in Fukui prefecture, Japan (Kamiya et al., will be published elsewhere). Microinjection of these microbes into T. castaneum was performed using a Nanoject II (Drummond Scientific Company) equipped with a glass needle having an outer tip diameter of ca. 25 lm except for the case of B. bassiana hyphal bodies, where a larger diameter of ca. 28 lm was adopted. 2.3. Sequences of genes and primers for qRT-PCR We used the following T. castaneum genes in this study: PPO1 (GLEAN_00325), PPO2 (GLEAN_14907), PPO3 (GLEAN_15848), Rel (GLEAN_11191), Attacin1 (Att1) (GLEAN_07737), Att2 (GLEAN_ 07738), Att3 (GLEAN_07739), Cecropin2 (Cec2) (GLEAN_00499), Cec3 (GLEAN_00500), Coleoptericin1 (Col1) (GLEAN_05093), Defnsin1 (Def1) (GLEAN_06250), Def2 (GLEAN_10517), Def3 (GLEAN_12469), and the normalizer of qRT-PCR, ribosomal protein L32 (RPL32) (GLEAN_06106). These sequences were retrieved from the Beetlebase (http://www.beetelebase.org), and primer pairs of respective mRNAs designed for qRT-PCR. The primer sequences for PPO1 and PPO2 are listed in Table 1. The primer pair for PPO2 was designed to amplify the identical fragment from both PPO2 and PPO3 cDNA templates. The other primer sequences appear in our previous papers (Yokoi et al., 2012a, 2012b). 2.4. Sequence analyses Amino acid sequence alignments were performed by the Clustal W program (Thompson et al., 1994) at DNA Data Bank of Japan web site (http://clustalw.ddbj.nig.ac.jp/index.php?lang=ja). Potential regulatory elements in the promoter regions of PPOs were searched by using a regulatory elements search algorithm, the ConSite (http://consite.genereg.net/) (Sandelin et al., 2004). 2.5. RNA extraction and qRT-PCR Total RNA was extracted from the whole body of T. castaneum and subjected to qRT-PCR analyses as described in our previous papers (Yokoi et al., 2012a, 2012b). Spectrophotometric scanning of RNA preparations showed that the ratios of A260/A280 and A260/A230 were always above 1.7 and 2.0, respectively. Since only the primer pairs for Att1, Att3, Cec2, Def3 and RPL32, span exon–intron boundaries among those used for qRT-PCR analyses, we digested contaminating genomic DNA prior to the first strand cDNA synthesis by utilizing a PrimeScript RT Reagent Kit with gDNA Eraser (TAKARA). One microgram of total RNA was used as a template for reverse transcription, and an aliquot of the first strand cDNA subjected to qRT-PCR-based mRNA determination using a SYBR Premix Ex Taq Perfect Real Time Kit Tli RNAaseH Plus (TAKARA) and a Thermal Cycler Dice Real Time System (Model TP800, TAKARA). The homogeneity of qRT-PCR products was confirmed by dissociation analyses. The threshold cycle numbers of the normalizer RPL32 determined by the second derivatives of the primary amplification curves were well consistent between

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K. Yokoi et al. / Journal of Invertebrate Pathology 132 (2015) 190–200

Table 1 Sequences of primers used for qRT-PCR. Target gene

Forward primer sequence (50 –30 )

Reverse primer sequence (50 –30 )

PPO1 PPO2

AGCTCGACAGTCAGGTGTTTG TTGGGCGTCTCCCTCAGCAA

GGTGCTCCTCGTCTAAATCTG TTGGGAATAAACAGCGAGAAGTT

Table 2 Sequences of primers for synthesizing T7 promoter-tagged cDNA. Target gene

Forward primer sequence (50 –30 )

Reverse primer sequence (50 –30 )

PPO1

TAATACGACTCACTATAGGGG-AACCGATTGGGGTGCAAATCG TAATACGACTCACTATAGGGG-TTGGGCGTCTCCCTCAGCAA TAATACGACTCACTATAGGGG-ATTGCTGCTGACGGGGGTTAT

TAATACGACTCACTATAGGG-GTGAATAAAGGACGGGAGGTC TAATACGACTCACTATAGGGG-ATTCGATGAAGGAGGGCAGGT TAATACGACTCACTATAGGGG-ATGTTCGGCATGATTTCACCTTT

PPO2 malE

T7 promoter sequences are in italics.

experiments. For detailed PCR conditions, see our previous paper (Yokoi et al., 2012a, 2012b). 2.6. RNAi RNAi-based gene knockdown was carried out basically as described in our previous papers (Yokoi et al., 2012a, 2012b). Sequences of primers used to prepare T7 RNA polymerase

NRFGE—-QANE NRFGE--DADE NRFKQ--DAEE NRFGD--DASE NRFGS--DAGR NRFGN--EATK NRFGDEEEVSR ***.. .. .

promoter-tagged cDNA templates of PPO1, PPO2, and a control, maltose binding protein E (malE) are shown in Table 2. The primer pair for PPO2 is located in the region of ORF completely shared by PPO2 and PPO3. The T7-tagged primer sequences for Rel are presented in our previous paper (Yokoi et al., 2012a). In brief, cDNA templates generated by conventional PCR with these primer pairs were used for dsRNA synthesis using a MEGAscript RNAi Kit (Ambion). One hundred nanograms of purified dsRNA typically in 32.2 nl of 10 mM Tris–HCl, pH 8.0 were injected into day 0 pupae, either singly or in combination, using a Nanoject II. Seventy-two hours later, the pupae were examined in terms of knockdown efficiency or further challenged with microbes.

2.7. Determination of PO activity PO activity was determined basically according to previous papers (Lee et al., 2000, 2002). As we had a technical difficulty in collecting clean pupal hemolymph, contents in the hemocoel, which included detached fluffy tissues in addition to hemolymph and hemocytes, were collected altogether by washing out dissected pupae with 50 ll of ice-cold 20 mM Tris–HCl, pH 8.0. This was immediately followed by the centrifugation at 2000 rpm for 10 min at 4 °C, and the supernatant taken as a hemolymph protein fraction. Protein concentration was determined with a Protein Assay Dye Reagent Concentrate (Bio-Rad) using bovine serum albumin (Sigma) as a standard. Fifty micrograms of hemolymph protein samples in 30 ll of 20 mM Tris–HCl, pH 8.0 was combined with 3 lg of curdlan (Wako Pure Chemicals) in 85 ll of the same buffer solution, and preincubated at 30 °C for 10 min. In the case

A

Tc-PPO1 Tm-PPO Tc-PPO2 Bm-PPO2 Bm-PPO1 Ms-PPO1 Ms-PPO2

48 49 48 50 50 50 50

59 57 56 58 58 58 60

B

Tc-PPO2 Tm-PPO Tc-PPO1 Ms-PPO1 Bm-PPO1 Ms-PPO2 Bm-PPO2

195 196 195 197 197 203 201

AYFREDLGINLHHWHWHLVYPFEAA-REVVAKNRRGELFYYMHQQIIARY AYFREDLGINLHHWHWHLVYPFEAA-REVVAKNRRGELFYYMHQQIIARY AYWREDIGLNLHHWHWHLVYPFEGA-REIVDKNRRGEIFYYMHQQIIARF AYFREDIGINLHHWHWHLVYPFDSADRSIVNKDRRGELFYYMHQQIIGRY AYFREDIGINLHHWHWHLVYPFDAADRAIVNKDRRGELFYYMHQQIIARY AYWREDLGINLHHWHWHLVYPFSASDEKIVAKDRRGELFFYMHQQIIARY AYWREDIGINLHHYHWHLVYPFTANDLSIVAKDRRGELFFYMHQQVIARF **.***.*.****.********..... .*.*.****.*.*****.*.*.

C

Tc-PPO2 Tm-PPO Bm-PPO1 Ms-PPO1 Tc-PPO1 Ms-PPO2 Bm-PPO2

355 356 358 358 355 360 358

NRTYYGDLHNMGHVFISYIHDPDHRHLESFGVMGDSATAMRDPIFYRWHSYIDDIFQ NRTFYGDMHNMGHVFISYVHDPDHRHLESFGVMGDSATAMRDPIFYRWHSYIDDIFQ NRPYYGDLHNMGHVFISYSHDPDHRHLEQFGVMGDSATAMRDPVFYRWHAYIDDIFH NRGYYGDLHNMGHVFAAYTHDPDHRHLEQFGVMGDSATAMRDPFFYRWHRFVDDVFN NRNYYGDFHNMGHILIGYIHDPDHRFLEPFGVMADPAVDLRDPLFFRWHAYIDDMFQ NRDLYGSIHNNMHSFSAYMHDPEHRYLESFGVIADEATTMRDPFFYRVHAWVDDIFQ NRELYGSIHNNGHSFTAYMHDPEHRYLEQFGVIADEATTMRDPFFYRWHAYIDDVFQ ** .**..**..*....*.***.**.** ***..*.*...***.*.*.*...**.*.

243 244 243 246 246 252 250

411 412 414 414 411 416 414

Fig. 1. Amino acid sequence alignment of conserved domains of insect PPOs. Amino acid sequences of PPOs from T. castaneum (Tc-PPO1 and Tc-PPO2), T. molitor (Tm-PPO, only one PPO identified in T. molitor), M. sexta (Ms-PPO1 and Ms-PPO2) and B. mori (Bm-PPO1 and Bm-PPO2) were carried out using the Clustal W algorithm. Alignments of proteolytic cleavage sites (A) and two independent Cu2+ binding sites (B and C) are shown. Amino acid residues shared by four or more proteins are shown by white letters on black background, while conservative amino acid substitutions are those on gray background. Asterisks on the bottom of the alignment denote amino acids shared by all the seven proteins while small dots indicate conserved or conservatively substituted amino acids at least in four proteins. A triangle in (A) indicate potential proteolytic cleavage site for PPO activation. Three each of larger dots in (B and C) indicate histidine residues that form coordination bond with Cu2+. Numbers on both ends of each line represent amino acid residue numbers of respective proteins. Amino acid sequences of Tm-PPO (JX987235), Ms-PPO1 (AF003253), Ms-PPO2 (AM293328), Bm-PPO1 (NM_001043870) and Bm-PPO2 (NM_001044069) were retrieved from the GenBank (http://www.ncbi.nlm.nih.gov/genbank/).

K. Yokoi et al. / Journal of Invertebrate Pathology 132 (2015) 190–200

0.3

more specific to PO, was used to estimate the substrate oxidation by contaminating hemocyte peroxidases (Giannoulis et al., 2007; Li et al., 1996; Mahbubur Rahman et al., 2007). To assess the degree of auto-oxidation of the substrate, the reaction mixture that lacked hemolymph proteins was prepared, and the changes of A468 monitored in a similar way.

0.2

2.8. Survival assay

Relative mRNA amount (/RPL32)

0.5

A

PPO1

naive malE

0.4

Rel

0.1

0 0

1

2

3

4

5

Pupal age in days 1

Relative mRNA amount (/RPL32)

193

PPO2

B

naive malE

0.8

Rel

0.6

0.4

0.2

0 0

1

2

3

4

5

Pupal age in days Fig. 2. Developmental changes of PPO1 and PPO2 mRNA in naïve and knockdown pupae. mRNA amounts of PPO1 (A) and PPO2 (B) were determined throughout the pupal stage of either naïve or Rel knockdown pupae that were treated with Rel dsRNA on day 0. MalE dsRNA injected pupae provided another control. The data of naïve day 0 pupae are shared by the two knockdown categories. Total RNA extraction and mRNA determination were done using a pool of three animals at each time point. Experiments were repeated independently at least three times, and the mean mRNA amounts relative to those of a normalizer, RPL32, are shown for respective samples. Vertical bars represent ±S.D.

Table 3 Potential NF-jB binding motifs found in upstream regions of T. castaneum PPO genes. Gene

Strand

jB motifs

Positiona

Inferred relevanceb

PPO1 PPO2 PPO3

(+) (+) (+)

GGGTATTCCC GGGGCTTACC GGGGATCTCC

282 to 291 48 to 57 1526 to 1515

IMD-Rel IMD-Rel IMD-Rel

a Since transcription initiation sites are not determined for these genes, positions of the binding motifs are shown relative to those for initiation ATG. b Inferred based on the results of Yokoi et al. (2012a).

of another coleopteran species, Holotrichia diomphalia, the PO activity in hemolymph is reported to be enhanced by curdlan (Lee et al., 1998). Then, 400 ll of 20 mM Tris–HCl, pH 8.0, containing saturated L-dopa substrate (Wako Pure Chemicals) and 5 mM CaCl2 were added, and the mixture kept at 30 °C. Absorbance at 468 nm of the reaction mixture, which represents the absorption peak of dopachrome, was thereafter measured at 0 (immediately after the addition of the substrate and CaCl2, without incubation at 30 °C), 5, 15, 30 and 60 min. In some experiments, phenylthiourea (Sigma) or tropolone (Sigma) was included in the reaction at the final concentration of 1.3 mM or 5 mM, respectively. Phenylthiourea functions as a competitive inhibitor of enzymatic oxidation of L-dopa by PO as well as by other oxidative enzymes (Barrett and Andersen, 1981; Ryazanova et al., 2012; Zhan et al., 2003). A metal ion chelator tropolone, the inhibition of which is

Survival assays with model bacterial pathogens were performed according to our previous papers (Yokoi et al., 2012a, 2012b). Briefly, day 0 pupae were treated with dsRNA of PPO1, PPO2, PPO1 plus PPO2 or control malE. After 72 h incubation at 30 °C, the pupae (day 3) were injected with 50 nl of either live E. cloacae or live B. subtilis using a Nanoject II. Life and death decisions of pupae were done by observing under the microscope the responses of pupae when touched by a thin and flexible plastic rod. The number of surviving pupae was counted every 24 h. Data were shown in Kaplan–Meier plots, and P-values calculated by Gehan–Breslow–Wilcoxon tests using a commercial software package (Ekuseru-Toukei 2010, Social Survey Research Information Co., Ltd.). The fungal pathogen B. bassiana was cultured on a SDY agar plate at 25 °C. The survival assays with B. bassiana were performed in two different ways, either by injecting cultured hyphal bodies into the pupal hemocoel or by immersing pupae in its conidium suspension. In these cases, animals were kept at 25 °C in order to allow adequate growth of the fungus, as well as to extend the pupal period since this fungal species exhibits relatively a slowkilling nature. Thus, day 0 pupae that had been reared at 30 °C were transferred to 25 °C just after dsRNA injection. Under these conditions the pupal period was typically extended to around 8.5 days at 25 °C from around 5.5 days at 30 °C. To prepare the hyphal body suspension, a piece of hypha from a SDY slant was inoculated into the same liquid medium and cultured for two days at 25 °C with continuous shaking. The culture was filtered through a syringe barrel stuffed with a piece of absorbent cotton, and the filtrate centrifuged. The precipitated cells were washed twice in sterile PBS, counted and diluted to 5  104 hyphal body cells/ml PBS. One hundred nanoliters of the freshly-prepared hyphal body suspension, equivalent to five hyphal body cells, were injected into day 3 control or knockdown pupae using a Nanoject II. Meanwhile, the conidia were collected from a fully-grown, dried culture plate and stored at 4 °C in the presence of a desiccant agent until use. The stored conidia were suspended in 0.02% Tween-80 before use, filtrated as above, and the density adjusted to 1  105 conidia/ml. Day 3 control or knockdown pupae were immersed in the conidium suspension for 1 min, and transferred into wells of a 24-well culture plate after removing excessive liquid with filter paper. In both types of fungal infection experiments, the pupae were kept in moist containers, and observed periodically. 2.9. Statistical analyses Statistical analyses were performed using the commercial software package (Ekuseru-Toukei 2010, Social Survey Research Information Co., Ltd.). 3. Results 3.1. Amino acid sequence alignment of PPOs of T. castaneum, Tenebrio molitor, Bombyx mori and Manduca sexta To clarify whether critical structures for zymogen activation and enzymatic activity were conserved in Tribolium PPO products, their deduced amino acid sequences were aligned with those of

194

K. Yokoi et al. / Journal of Invertebrate Pathology 132 (2015) 190–200

Relative mRNA amount (/RPL32)

0.16

A

PPO1

0.14 0.12 0.1

*

0.08

* *

*

0.06 0.04 0.02 0

Uc PBS Ec Ml 1h

Sc

Uc PBS Ec Ml 3h

Sc

Uc PBS Ec Ml 6h

Sc

Uc PBS Ec Ml 24h

Sc

Hours after treatment

Relative mRNA amount (/RPL32)

0.8 0.7

PPO2

B

0.6 0.5 0.4 0.3 0.2

0

* *

* * *

0.1

* * * Uc PBS Ec Ml 1h

Sc

Uc PBS Ec Ml 3h

Sc

Uc PBS Ec Ml 6h

Sc

Uc PBS Ec Ml 24h

Sc

Hours after treatment Fig. 3. Changes of PPO mRNA upon challenges with heat-killed microbes. Day 3 pupae were injected with PBS suspensions of heat-killed E. coli (Ec), M. luteus (Ml) or S. cerevisiae (Sc). Unchallenged (Uc) and PBS-injected (PBS) pupae were used as controls. The mRNA amounts of PPO1 (A) and PPO2 (B) relative to those of RPL32 were then monitored at one, three, six and 24 h post microbial challenges. mRNA determination after respective treatments was done using a pool of three animals at each time point. Experiments were repeated independently at least three times, and bars represent means with S.D. Asterisks indicate significantly different values from Uc control pupae at the same time points (P < 0.05 by Student’s t-test).

well-characterized PPOs from T. molitor (Lee et al., 1999), B. mori (Kawabata et al., 1995) and M. sexta (Hall et al., 1995; Jiang et al., 1997). The alignments of three distinct regions are shown in Fig. 1. The alignments revealed that both the proteolytic cleavage sites (Fig. 1A) and the two Cu2+ binding sites (Fig. 1B and C) were highly homologous among these proteins, with the complete conservation of two critical residues, Arg and Phe for proteolytic cleavage as well as of three indispensable His residues for the each Cu2+ binding site. These results suggest that both T. castaneum PPO1 and PPO2 encode functional PPO proteins. 3.2. Changes in mRNA amounts of the two PPOs during the pupal development Changing profiles of PPO1 and PPO2 mRNA during pupal stage were determined by qRT-PCR. The expression pattern of these genes in pupae has already been demonstrated semi-quantitatively in addition to those in pre-pupae and adults (Arakane et al., 2005). Here, we presented more minute changing profiles of these mRNAs in naïve as well as Rel and control knockdown pupae in parallel (Fig. 2). The amounts of PPO1 mRNA in naïve pupae that was relatively abundant on day 0 declined continuously and reached the lowest levels on day 4 and day 5 (Fig. 2A). In contrast, while the PPO2 mRNA level was similar to that of PPO1 on day 0, it thereafter went up, peaked on day 2, then declined to the lowest levels on

day 4 and day 5 (Fig. 2B). We also examined the possible regulation of these two PPO genes by a NF-jB transcription factor during the pupal development. We previously investigated the involvement of NF-jB transcription factors, namely Dif1/Dif2 and Rel, in microbial induction of T. castaneum AMP genes, and presented inferred binding motifs for respective NF-jB transcription factor classes via in silico analyses on the regulatory region of the AMP genes (Yokoi et al., 2012a). Similarly, we searched potential jB motifs in the upstream regulatory regions of T. castaneum PPO genes by utilizing the Consite algorithm (Sandelin et al., 2004). Table 3 shows potential NF-jB binding motifs found in the 50 regions of PPO1 and PPO2 as well as PPO3. These motifs fall in the category of putative Rel binding motifs (Yokoi et al., 2012a), suggesting the regulation by the IMD-Rel pathway. We assessed the role of Rel in constitutive PPO1 and PPO2 expression by using RNAi-based gene knockdown approaches. As shown in Fig. 2, Rel knockdown as well as control malE knockdown did not seem to affect the developmental kinetics of PPO1 and PPO2 when compared to that of naïve pupae, indicating that Rel is not a key factor for the constitutive expression of these PPO genes. 3.3. PPO1 and PPO2 mRNA levels after microbial challenge In addition to the well-established cases of AMP genes, the expression of some immunity-related genes is frequently modulated by immune challenge (Altincicek et al., 2013; De Gregorio et al., 2001;

Relative mRNA amount (/RPL32)

Relative mRNA amount (/RPL32)

K. Yokoi et al. / Journal of Invertebrate Pathology 132 (2015) 190–200

0.06

A

PPO1

0.05 0.04 0.03 0.02 0.01 0 dsRNA 0.8 0.7

malE

*

*

PPO1

PPO1+PPO2

B

PPO2

0.6

using two model bacterial pathogens, Gram-negative E. cloacae and Gram-positive B. subtilis, both of which possess DAP-type peptidoglycan. We first examined the gene knockdown efficiency for the two PPO genes. As shown in Fig. 4, sufficient reduction of the mRNA amount ranging from 89% to 94% was observed for both PPO1 and PPO2 upon single and combined knockdown at 72 h post dsRNA treatment. Subsequently, the knockdown pupae were challenged with live E. cloacae or B. subtilis, and survival was monitored every 24 h. When challenged by E. cloacae, survival rates of single or double PPO knockdown pupae did not differ significantly from that of control pupae (Fig. 5A). The single knockdown of either PPO1 or PPO2 seemed to somewhat weaken survival upon B. subtilis challenge, but the differences were not significant: P = 0.196 and 0.167 for PPO1 and PPO2 knockdown, respectively (Fig. 5B). On the other hand, double knockdown pupae of PPO1 and PPO2 succumbed to B. subtilis significantly faster than control pupae with a P-value of 0.015 (Fig. 5B). 3.5. PPO gene knockdown and hemolymph PO activities

0.5 0.4 0.3 0.2

*

0.1 0 dsRNA

195

malE

PPO2

* PPO1+PPO2

Fig. 4. Knockdown of PPO1 and PPO2 by dsRNA injection. Day 0 pupae were injected with PPO dsRNA either singly or in combination. Pupae treated with malE dsRNA served as controls. Seventy-two hours later, the mRNA levels of PPO1 and PPO2 in respective samples were determined relative to those of RPL32 using a pool of three pupae. Each bar represents a mean with S.D. from three independent experiments. Asterisks indicate significantly different values from those of malE-treated controls (P < 0.05 by Student’s t-test).

Zou et al., 2007). Here, to examine whether or not T. castaneum PPOs show such trend, we injected heat-killed E. coli (Gram-negative bacterium bearing DAP-type peptidoglycan), M. luteus (Gram-positive bacterium bearing Lys-type peptidoglycan), S. cerevisiae (budding yeast bearing b-1,3 glucan), or vehicle PBS as a control into day 3 naïve pupae, and measured the PPO1 and PPO2 mRNA amounts in the whole body at 1, 3, 6 and 24 h post treatment (Fig. 3). Zou et al. (2007) have reported that the mRNA levels of these PPO were not heavily affected by microbial challenge in adult beetles at 24 h post challenge. The PPO1 mRNA amount in day 3 pupae at 1 h post injection of the three microbes or PBS were similar to the unchallenged level, and did not change appreciably by 6 h in comparison with unchallenged controls. The mRNA amount of PPO1 elevated significantly over the unchallenged control at 24 h after the bacterial injections (Fig. 3A). The PPO1 mRNA levels also seemed to increase upon challenge with the budding yeast, but the difference was not statistically significant (Fig. 3A). The unchallenged levels of PPO1 mRNA decreased gradually during this period of 24 h, which was consistent with the pattern shown in Fig. 2A. In contrast, the mRNA amount of PPO2 basically decreased after the microbial challenges at 3, 6 and 24 post challenge in comparison with the unchallenged control (Fig. 3B). The mRNA amount after microbial challenges bottomed out at around 6 h with values corresponding to ca. 5% of the unchallenged control. Thus, the two PPO genes exhibited different expression patterns in response to microbial challenge, and the patterns did not seem to be influenced by the microbial species used, which bear distinct pathogen-associated molecular patterns. 3.4. PPO genes and defense against bacterial pathogens To investigate whether these PPO genes of T. castaneum have a role in antibacterial host defense, we conducted survival assays

Subsequently, we compared PO activities between double PPO knockdown pupae and malE dsRNA-treated control pupae to elucidate whether the decreased viability by the knockdown was associated with altered PO activity. We managed to prepare a hemolymph protein fraction (see Section 2.7), and measured PO activity using L-dopa as a substrate. First, we examined how much of an increase of A468, which corresponds to the absorption peak of dopachrome, was ascribable to PO activity using untreated day 3 pupae (Fig. 6A). A reaction mixture lacking hemolymph proteins was also prepared to assess the auto-oxidation of the substrate with time, and the results revealed that an appreciable part of the A468 increment of control reaction that contained both the substrate and hemolymph proteins was due to the substrate autooxidation. The increase of A468 over the substrate auto-oxidation, which was referable to the components of the hemolymph protein fraction, was investigated further by utilizing phenylthiourea and tropolone. While the tendencies were somewhat obscure in the early phase of incubation, it became evident at 30 and 60 min that the increment of A468 was effectively suppressed to the same extent by the both inhibitors. This suggests that the substrate oxidation associated with the hemolymph protein fraction was exerted mainly by PO rather than by other potential oxidative enzymes. Although A468 in the presence of the inhibitors at 30 and 60 min were still significantly higher than those by autooxidation, we did not investigate this issue further. Then, we proceeded to PO activity determination of double knockdown and control pupae. The results were depicted in Fig. 6B, where differences of A468 values from those by auto-oxidation at the corresponding time points were conveniently taken as PO activities. The double knockdown pupae exhibited a lowered profile than that of malE-treated control. Thus, the double knockdown of PPO genes indeed suppressed the PO enzymatic activity, possibly leading to the weakened defense phenotype against B. subtilis. 3.6. AMP gene induction in double PPO knockdown pupae upon B. subtilis challenge We have previously shown that IMD or Rel knockdown, which results in considerably reduced bacterial induction of the IMD-pathway dependent AMP genes, severely impairs host defense against both E. cloacae and B. subtilis (Yokoi et al., 2012a, 2012b). To test whether the double PPO knockdown influenced such AMP gene induction, we measured the mRNA amounts of all the nine T. castaneum AMP genes at 6 h post B. subtilis challenge (Fig. 7). We did not determine the unchallenged mRNA levels of these AMP genes in this experiment. For the characteristics of bacterial induction of each

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Fig. 5. PPO gene knockdown and antimicrobial host defense. Day 0 pupae were treated with PPO1 and PPO2 dsRNA either singly or doubly. The pupae were injected with live E. cloacae (A), B. subtilis (B), or live hyphal bodies of B. bassiana (C) or treated with B. bassiana conidium suspension (D) at 72 h post dsRNA treatment. Thereafter, the numbers of surviving pupae were counted periodically. Experiments were independently repeated at least three times with 8–10 animals, and the sums of the data are presented in Kaplan–Meier plots. dsRNA treatments that resulted in statistically different survival rates from that of the malE-treated control by Gehan–Breslow–Wilcoxon analysis (P < 0.05) are marked with asterisks. For detailed infection conditions, see Section 2.8.

AMP gene, refer to our previous paper (Yokoi et al., 2012b). The induced mRNA levels of all AMP genes but Def1 were not affected significantly by the background of double PPO knockdown or malE control knockdown. Def1 is classified into group IV AMP genes that show negligible or very weak induction in pupae upon microbial challenge (Yokoi et al., 2012b), as well as in larvae and adults (Kitamoto et al., our unpublished observation). Indeed, the induced mRNA levels of Def1 by B. subtilis infection, as well as those of another group IV AMP gene Att3, are extremely lower than those of the other seven AMP genes (Fig. 7). Therefore, we are skeptical about the immunological significance of this slightly higher mRNA level of Def1 found in the double PPO knockdown pupae compared to the control, while we cannot exclude possible roles of this phenomenon. Thus, we concluded that the observed lesser survival of double PPO knockdown pupae upon B. subtilis infection (Fig. 5B) did not result from the unexpected modulation of another major effector mechanism, induction of AMP genes. 3.7. PPO genes and defense against fungal infection Finally, we tested the roles of PPOs in antifungal host defense using an entomopathogenic fungus, B. bassiana. We employed two distinct methods of infection, injecting cultured hyphal bodies intrahemocoelically or immersing animals in conidium suspension, the latter of which mimics the natural way of B. bassiana infection. As regards the direct hyphal body injection, we found that it was highly virulent to T. castaneum pupae. For example, pupae consistently died by 48–72 h irrespective of knockdown types when hyphal body suspension equivalent to 50–500 cells were injected (data not shown). Therefore, we here employed inoculation of a

much smaller dose, equivalent to five hyphal body cells per pupa, which killed pupae more slowly. In this experiment, we simply injected 100 nl of hyphal body suspension with a density of 5  104 cells/ml PBS, but did not actually affirm exact numbers of cells injected into each animal. The results of hyphal body injection are depicted in Fig. 5C. Respective knockdown seemingly reduced the survival rate, but the survival of PPO2 knockdown pupae did not differ significantly from that of malE control: P-values were 0.007 (PPO1 knockdown), 0.111 (PPO2 knockdown) and 0.013 (double PPO knockdown), respectively. On the other hand, when immersion in conidium suspension was employed, all experimental knockdown weakened moderately but significantly host defense in comparison with control knockdown, with P-values of 0.013 (PPO1 knockdown), 0.031 (PPO2 knockdown) and 0.048 (double PPO knockdown) (Fig. 5D). The immersion methods took more time to kill animals compared to the direct injection of hyphal bodies probably because it requires the steps of conidium germination and the following exoskeletal penetration by hyphae. Thus, each of single PPO knockdown, except the PPO2 knockdown followed by the hyphal body injection, was shown to impair the beetle’s antifungal host defense to some extent, while the double PPO knockdown did not exhibit additive or synergistic effects. 4. Discussion In this study, we characterized the two PPO genes of T. castaneum and investigated their roles in antimicrobial host defense. We first carried out amino acid sequence analyses in comparison with well-characterized PPOs from other insect species. Crucial motifs for the proteolytic zymogen activation and Cu2+ binding,

K. Yokoi et al. / Journal of Invertebrate Pathology 132 (2015) 190–200

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Incubation time (min) Fig. 6. PO activity and effects of PPO1 and PPO2 double knockdown. (A) Hemolymph protein fractions were prepared from untreated day 3 pupae, combined with a substrate L-dopa, and the changes of A468, which represents the oxidation of the substrate, were measured with time. The measurement was also performed in the presence of either phenylthiourea or tropolone, while the reaction in the absence of hemolymph proteins gave the degree of non-enzymatic auto-oxidation of the substrate. Hemolymph proteins were prepared from four to five pupae each time, and the experiments were independently repeated three times. Data are presented as means from three independent determinations with S.D., and the mean values at 0 min are set to 0 for respective experimental categories. Points with the same letter are not statistically different (Tukey’s multiple comparison test for each time point, P > 0.05). (B) Day 0 pupae were treated with either PPO1 plus PPO2 dsRNA or malE dsRNA, and hemolymph protein fractions prepared 72 h later from four to five pupae. The oxidation of L-dopa (A468) in the presence of the hemolymph proteins was determined as in (A). Auto-oxidation of the substrate was also measured, and A468 values of auto-oxidation at respective time points were subtracted from the corresponding values obtained with hemolymph protein samples. The resulting A468 values at 0 min after the subtraction were set to 0 for both double PPO knockdown and control knockdown categories, and the subsequent A486 increment over auto-oxidation levels conveniently taken as PO activities. Experiments were independently repeated three times, and data are presented as means with S.D. Statistically different values from malE-treated controls of the corresponding time points are marked with asterisks (P < 0.05 by Student’s t-test).

both of which are indispensable for inducible melanin synthesis (Cerenius and Söderhäll, 2004), were highly conserved as well in T. castaneum counterparts, suggesting that the PPO genes in the genome of T. castaneum encode functional PPO proteins. Then, we examined the expression levels of T. castaneum PPO genes in the pupal stage, at which we have been studying immunity-related genes and defense phenotypes of this model beetle (Koyama et al., 2015; Yokoi et al., 2012a, 2012b). The mRNAs of both PPO1 and PPO2 were readily detectable the in pupae, indicating that the two genes were indeed expressed in this developmental stage. Previously, Arakane et al. (2005) have documented the expression and changing patterns of the two PPO mRNAs while these authors were in search for a gene responsible for cuticle tanning, laccase 2. They employed gel analyses of RT-PCR products,

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and showed that the two PPO mRNAs are relatively abundant in prepupae as well as in middle stage pupae, decline toward adult emergence. The expression of PPO genes in the adult stage is also reported by Zou et al. (2007). We examined more detailed developmental changes in the pupal stage by using qRT-PCR analyses and found that the two mRNAs actually showed distinct changing profiles while the low levels of both mRNAs at later phase of pupal development were consistent with the previous study: the PPO1 mRNA consistently went down during pupal development; the PPO2 mRNA peaked in the middle of the stage. The differences of developmental profiles may suggest their functional differences. In addition, we demonstrated that these distinct, constitutive expression patterns of PPO1 and PPO2 in pupae did not depend on Rel function. Meanwhile, the expression of PPO genes in the larval stage has been reported in a recent paper by Behrens et al. (2014). The results of expression analyses by others and us collectively indicate that the PPO genes of T. castaneum exhibit developmental stage-dependent expression patterns, and this also holds true in Drosophila (Asano and Takebuchi, 2009). Generally, PPO expression are confined to specific types of hemocytes, such as oenocytoids of lepidopteran insects (Ashida et al., 1988; Iwama and Ashida, 1986; Jiang et al., 1997) or crystal cells of Drosophila (Williams, 2007). The elevated PO activity observed upon infection is considered to be regulated by biochemical processes rather than by transcriptional ones since experimental pathogen infection usually does not induce PPO genes themselves (De Gregorio et al., 2001) whereas many other genes coding for the constituents of PPO activating cascade are upregulated (De Gregorio et al., 2002; Ligoxygakis et al., 2002). When we injected heat-killed microbial suspension into day 3 pupae, the PPO2 mRNA level measured in whole body declined and was almost depleted by 6 h post injection, while that of PPO1 was relatively unaffected by this time point. Interestingly, the changing profiles of the two PPO mRNAs upon microbial challenge did not depend on microbial species while the three model microbes that we used bear distinct pathogen-associated molecular patterns. The different responses of the two PPO genes upon microbial challenge may suggest different sites of expression as well as different function of the two genes. PPO proteins usually lack a signal peptide (Cerenius and Söderhäll, 2004), and thus their secretion into the hemolymph is accompanied by cell rupture/death (Bidla et al., 2007). The observed drastic decline of T. castaneum PPO2 mRNA upon microbial injection may reflect the lysis of a certain cell type specific to PPO2 expression. A similar appreciable decline of PPO mRNA post microbial challenge is also reported in another coleopteran species, T. molitor (Dobson et al., 2012). On the other hand, our results may also suggest that PPO1 expression, which was much less sensitive to microbial injection, takes place in distinct, more stable cell/hemocyte types from those for PPO2 expression. Whilst more cytological studies will be needed, this possible spatiotemporally different expression patterns suggest different roles between the two PPO proteins of T. castaneum. In the meantime, we do not exclude the possibility that the two PPO genes are transcriptionally regulated in different manners upon infection. The indispensability of melanization by PO in host defense has long been the subject of debate (Cerenius et al., 2008). While many researchers have taken melanization reaction as an important constituent of invertebrate innate immune systems, there are indeed several studies open to question in terms of PO involvement in host defense. In some studies using non-model insects, the measured PO activity did not correlate well with observed defense phenotypes of individuals (Reviewed in Gonzalez-Santoyo and Cordoba-Aguilar (2012)). As well in recent studies using genetically manipulated model insect species, namely Drosophila melanogaster and Anopheles gambiae, conclusions vary depending on respective combinations of insects and pathogen species, and tar-

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Fig. 7. AMP gene induction of double PPO knockdown pupae in the early phase of B. subtilis infection. PPO1 and PPO2 double knockdown pupae (PPO) or malE dsRNA-injected control pupae (malE) were injected with a live suspension of B. subtilis as in Fig. 6B, and the mRNA levels of all the nine AMP genes in the genome of T. castaneum were determined at 6 h post challenge. Each AMP gene name is shown along with its inferred regulation, via the Toll, the IMD or both pathways, and AMP genes that are not or only very weakly induced by microbial challenge in pupal stage are marked as ‘none’ (Yokoi et al., 2012b). Experiments were independently repeated three times, and bars represent means with S.D. The ordinate axe is on a logarithmic scale. ⁄ Significantly different from malE control (P < 0.05 by Student’s t-test).

geted genes (Leclerc et al., 2006; Schnitger et al., 2007; Tang et al., 2006; Yassine et al., 2012). All these studies targeted genes coding for upstream protease components of PPO activating cascade or an opsonin. Since insect immune signaling pathways are likely to be highly branched and networked, the removal of these upstream components could possibly influence both known and undefined effector mechanisms besides PPO activation, which may result in seemingly inconsistent outcomes. More recently, Lemaitre and coworkers have generated Drosophila lines lacking crystal cell–associated two PPO genes among the three in the fly genome. By utilizing the animals that carry deletions in these genes encoding the terminal components of the PPO activation cascade, they have unequivocally shown the indispensability of melanization reaction in antimicrobial host defense of Drosophila (Binggeli et al., 2014). We also directly knocked down the two PPO genes of T. castaneum in this study. We performed dsRNA injection with day 0 pupae and confirmed efficacious knockdown of the PPO genes on day 3 at the mRNA level. We did not examine the knockdown at the protein level. Instead, we compared PO activities between control and knockdown animals by preparing hemolymph protein fractions. Whereas we could not perform minute enzyme kinetics experiments because of insufficient qualities of our hemolymph protein preparation, at least we were able to show that the double PPO knockdown indeed resulted in decreased PO activities. While we do not rule out the idea that moderate phenotype changes by the knockdown can be ascribed to moderate decline of PPO zymogens, some configurations of targeted PPO genes and microbial species impaired host defense in a statistically significant manner. The double knockdown of PPO1 and PPO2 reduced significantly survival of pupae against injected B. subtilis but not against E. cloacae, while single knockdown of either PPO1 or PPO2 did not change greatly the survival rates. These results are consistent with those in Drosophila that lacks two PPO genes (Binggeli et al., 2014). Furthermore, when challenged with B. subtilis, double PPO knockdown did not affect the induced mRNA levels of all the nine AMP genes of T. castaneum except Def1 belonging to group IV AMP genes that show negligible or very weak induction upon microbial challenge. Taken together, our results suggest that the importance of PO activities in

host defense of T. castaneum differs depending on invading bacterial species, and that the products of two PPO genes exert a somewhat redundant role in host defense against B. subtilis in the hemocoel of T. castaneum. Similarly, when we challenged knockdown pupae with B. bassiana by injecting the hyphal bodies, single knockdown of PPO1 as well as double PPO knockdown resulted in modest but significant decline of the survival rate. By contrast, single PPO2 knockdown seemed to be less effective. These results may suggest that PPO1 contributes more to eliminate B. bassiana hyphal bodies and/or hyphae or to suppress its growth in the hemocoel of T. castaneum than PPO2 does. On the other hand, when we employed the infection via immersion in the conidium suspension, both of the single PPO knockdown impaired survival of pupae moderately, and the combined knockdown did not enhance further the suppression of host defense phenotype. Since PPO2 seemed not to function as effective as PPO1 in terms of defense against B. bassiana hyphal bodies and/or hyphae in the hemocoel, the observed contribution of PPO2 upon natural conidium infection might be associated with a function of its product exerted in the pupal integument. In B. mori, it has been demonstrated that the larval cuticle contains PPO proteins as well as its activating cascade components (Ashida and Brey, 1995), and that these PPO proteins have an oenocytoid origin and are transepithelially transported to and stored in the cuticle (Asano and Ashida, 2001). Although we do not know the cuticular occurrence of PPO proteins in the beetle, it might be possible that PPO2 protein is mainly functioning to prevent the penetration of elongating hyphae of B. bassiana in the cuticle while PPO1 protein plays a larger role in the hemocoel. The important roles of both PPO1 and PPO2 in defense against B. bassiana infection has also been demonstrated in Drosophila, and these authors document the spatiotemporally different properties of the two PPO proteins: both of them have a crystal cell origin, but PPO1 is probably secreted into the hemolymph while PPO2 stays in crystal cells; PPO1 function in an immediate phase of infection or injury, which is followed by the function of PPO2 (Binggeli et al., 2014). We consider that it is as well possible that the two T. castaneum PPOs have different localization and function although more direct evidence

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will be needed. In lepidopteran species such as M. sexta, two PPO genes are considered to be expressed coordinately in oenocytoids, and a heterodimer of the two distinct polypeptides comprises a major molecular species of PPO proteins in hemolymph (Jiang et al., 1997; Li et al., 2009). On the other hand, since T. castaneum PPO1 and PPO2 exhibited distinct developmental kinetics, different expression upon microbial infection, and apparently differential contributions to antimicrobial host defense, it may be interesting to consider that these T. castaneum PPO proteins function as a homodimer of either PPO1 or PPO2 polypeptide. In summary, we characterized the two PPO genes and their products of the model beetle, T. castaneum. The two PPO genes were supposed to encode functionally active zymogens, and the double knockdown resulted in decreased enzymatic activity in the hemolymph. Developmental kinetics and microbial induction profiles of the two PPO genes in the pupal stage suggested their differences in spatiotemporal expression, as well as differences in localization and function of the products. RNAi-mediated gene knockdown demonstrated importance of PPO genes in host defense of T. castaneum against B. subtilis and B. bassiana, and the two PPO genes were supposed to exert distinct roles upon infection of B. bassiana. Thus, the present study has added a new example that delineates the importance of PPO genes in antibacterial and antifungal host defense in a coleopteran insect species. As mentioned above, the PPO genes of T. castaneum display differential expression depending on developmental stages. This suggests that major effector mechanisms functioning to eliminate invading microbial pathogens might vary in a developmental stage-dependent manner. In this connection, we are currently investigating the developmental expression and microbial induction of immunity-related genes of this model beetle (Kitamoto et al., unpublished). However, the immune defense system could be more diverse than we expect even in a single insect species, as demonstrated by a recent study that suggests a differential usage of distinct immune effector mechanisms by T. castaneum larvae of two independent populations upon oral infection of an entomopathogen B. thuringiensis (Behrens et al., 2014). Our present and future studies on this model beetle species, in conjunction with those by others, hopefully will provide useful information regarding the development of control methods for notorious coleopteran pests, such as Leptinotarsa decemlinaeata (Campbell et al., 2010). Acknowledgements We thank Dr. Y. Yagi for the generous gift of E. cloacae and B. subtilis, and Dr. T. Ushimaru for S. cerevisiae S288S. We also thank Drs. T. Shinoda, A. Miyanoshita and M. Murata for providing T. castaneum. This work was supported in part by JSPS KAKENHI Grant Numbers 23658047 and 25450486 to KM. References Altincicek, B., Elashry, A., Guz, N., Grundler, F.M., Vilcinskas, A., Dehne, H.W., 2013. Next generation sequencing based transcriptome analysis of septic-injury responsive genes in the beetle Tribolium castaneum. PLoS ONE 8, e52004. Arakane, Y., Muthukrishnan, S., Beeman, R.W., Kanost, M.R., Kramer, K.J., 2005. Laccase 2 is the phenoloxidase gene required for beetle cuticle tanning. Proc. Natl. Acad. Sci. U.S.A. 102, 11337–11342. Asano, T., Ashida, M., 2001. Transepithelially transported pro-phenoloxidase in the cuticle of the silkworm, Bombyx mori. Identification of its methionyl residues oxidized to methionine sulfoxides. J. Biol. Chem. 276, 11113–11125. Asano, T., Takebuchi, K., 2009. Identification of the gene encoding prophenoloxidase A(3) in the fruitfly, Drosophila melanogaster. Insect Mol. Biol. 18, 223–232. Ashida, M., Brey, P.T., 1995. Role of the integument in insect defense: pro-phenol oxidase cascade in the cuticular matrix. Proc. Natl. Acad. Sci. U.S.A. 92, 10698– 10702. Ashida, M., Ochiai, M., Niki, T., 1988. Immunolocalization of prophenoloxidase among hemocytes of the silkworm, Bombyx mori. Tissue Cell 20, 599–610.

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