Comparative genomic analysis of the Tribolium immune system
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Tribolium castaneum is a species of Coleoptera, the largest and most diverse order of all eukaryotes. Components of the innate immune system are hardly known in this insect, which is in a key phylogenetic position to inform us about genetic innovations accompanying the evolution of holometabolous insects. We have annotated immunity-related genes and compared them with homologous molecules from other species.
Around 300 candidate defense proteins are identified based on sequence similarity to homologs known to participate in immune responses. In most cases, paralog counts are lower than those of Drosophila melanogaster or Anopheles gambiae but are substantially higher than those of Apis mellifera. The genome contains probable orthologs for nearly all members of the Toll, IMD, and JAK/STAT pathways. While total numbers of the clip-domain serine proteinases are approximately equal in the fly (29), mosquito (32) and beetle (30), lineage-specific expansion of the family is discovered in all three species. Sixteen of the thirty-one serpin genes form a large cluster in a 50 kb region that resulted from extensive gene duplications. Among the nine Toll-like proteins, four are orthologous to Drosophila Toll. The presence of scavenger receptors and other related proteins indicates a role of cellular responses in the entire system. The structures of some antimicrobial peptides drastically differ from those in other orders of insects.
A framework of information on Tribolium immunity is established, which may serve as a stepping stone for future genetic analyses of defense responses in a nondrosophiline genetic model insect.
KeywordsAdditional Data File Hemocyte Holometabolous Insect Family Expansion Serpin Gene
- β GRP
Gram-negative binding protein
peptidoglycan recognition protein
proPO activating factor
reactive nitrogen species
reactive oxygen species
noncatalytic serine proteinase homolog
Tribolium beetles harbor a range of natural pathogens and parasites, from bacteria to fungi, microsporidians and tapeworms [1, 2]. There is good evidence for genetic variation in resistance to the tapeworm and a linked cost of resistance in terms of growth and reproduction . Cross-generational transfer of immune traits  may occur in Tenebrio molitor, a close relative of Tribolium castaneum. RNA interference experiments demonstrate that Tribolium laccase-2 is responsible for cuticle pigmentation and sclerotization . While these observations are interesting, our knowledge of the genetic constituents of Tribolium immunity is almost blank at the cellular and molecular levels, in contrast to the vast amount of information regarding Drosophila melanogaster and Anopheles gambiae defense responses [6, 7]. Given the high efficiency of RNA interference and powerful tools of molecular genetics , it is particularly appealing to use T. castaneum for the dissection of insect immune pathways. Acquired knowledge may be useful in controlling beetle pests that feed on crop plants or stored products.
In the broader field of beetle immunity, research has been focused mainly on two effector mechanisms, namely antimicrobial peptide synthesis and prophenoloxidase (proPO) activation . Defensins, coleoptericins, cecropin and antifungal peptides have been isolated from coleopteran insects and characterized biochemically [10, 11, 12]. A homolog of human NF-κB (Allomyrina dichotoma Rel A) up-regulates the transcription of a coleoptericin gene . Active phenoloxidase generates quinones for melanin formation, wound healing, and microbe killing. ProPO activation has been investigated in Holotrichia diomphalia [14, 15, 16]. ProPO activating factor 1 (Hd-PPAF1) cleaves proPO to generate active phenoloxidase in the presence of Hd-PPAF2, the precursor of which is activated by Hd-PPAF3 via limited proteolysis. While all these PPAFs contain an amino-terminal clip domain, PPAF2 (in contrast to PPAF1 or PPAF3) does not have catalytic activity since its carboxy-terminal serine proteinase-like domain lacks the active site serine. A 43 kDa inhibitor down-regulates the melanization response in H. diomphalia .
To date, components of the innate immune system are hardly known in T. castaneum and neither is it clear how they differ from homologous molecules in the honeybee, mosquito or fruitfly [6, 7, 18]. This lack of knowledge does not seem to reconcile with the critical phylogenetic position of this coleopteran species, which should inform us a lot about genetic variations in the evolution of holometabolous insects. Information regarding defense responses in T. castaneum, a member of the largest and most diverse order of eukaryotes, is highly desirable for the biological control of crop pests and disease vectors. Consequently, we have used its newly available genome assembly to annotate immunity-related genes and analyze their phylogenetic relationships with homologous sequences from other insects. In this comparative overview of the Tribolium defense system, we describe plausible immune pathway models and present information regarding the molecular evolution of innate immunity in holometabolous species.
Results and discussion
Overview of the Triboliumimmune system
T. castaneum has a sizable repertoire of immune proteins predicted to participate in various humoral and cellular responses against wounding or infection (Additional data file 1). Like other insects [6, 7, 19], cuticle and epithelia lining its body surfaces, tracheae and alimentary tract may serve as a physiochemical barrier and local molecular defense by producing antimicrobial peptides and reactive oxygen/nitrogen species (ROS/RNS). While this line of defense may block most pathogens, others enter the hemocoel where a coordinated acute-phase reaction could occur to immobilize and kill the opportunists. This reaction, including phagocytosis, encapsulation, coagulation and melanization, is probably mediated by hemocytes and molecules constitutively present in the circulation. These first responders may not only control minor infections but also call fat body and hematopoietic tissues for secondary responses if necessary. At the molecular level, the following events should take place in all insects, including the beetle: recognition of invading organisms by plasma proteins or cell surface receptors, extra- and intracellular signal transduction and modulation, transcriptional regulation of immunity-related genes, as well as controlled release of defense molecules.
Multiple sequence alignment suggests that β-1,3-glucan-recognition proteins (β GRPs) and Gram-negative binding proteins (GNBPs) are descendents of invertebrate β-1,3-glucanases . Lacking one or more of the catalytic residues, these homologous molecules do not possess any hydrolytic activity. They are widespread in arthropods and act in part to recognize microbial cell wall components such as β-1,3-glucan, lipoteichoic acid or lipopolysaccharide. We have identified three β GRPs in T. castaneum. Tc-β GRP1 and AgGNBP-B1 through -B5 are closely related and represent a young lineage, whereas Tc-β GRP2 and Tc-β GRP3 belong to an ancient group that arose before the radiation of holometabolous insects (Additional data file 2). Since Drosophila has no β GRP-B and Anopheles has five, the presence of a single gene (encoding Tc-β GRP1) in the beetle can be useful for elucidating function of this orthologous group. In addition to the glucanase-like domain, members of the second group contain an amino-terminal extension of about 100 residues. In Bombyx mori β GRP, this region recognizes β-1,3-glucan also . M. sexta β GRP2 binds to insoluble β-1,3-glucan and triggers a serine proteinase cascade for proPO activation .
C-type lectins (CTLs) comprise a wide variety of soluble and membrane-bound proteins that associate with carbohydrates in a Ca2+-dependent manner . Some insect CTLs recognize microorganisms and enhance their clearance by hemocytes . Gene duplication and sequence divergence, particularly in the sugar-interacting residues, lead to a broad spectrum of binding specificities for mannose, galactose and other sugar moieties. These proteins associate with microbes and hemocytes to form nodules  and stimulate melanization response . T. castaneum encodes sixteen CTLs: ten (Tc-CTL1, 2, 4 through 10, and 13) with a single carbohydrate recognition domain and one (Tc-CTL3) with two. Five other proteins, tentatively named Tc-CTL11, 12, 14, 15 and 16, contain a CTL domain, a transmembrane region (except for Tc-CTL11), and other structural modules: CTL11 has three CUB and three EGF; CTL12 has six Ig and three FN3; CTL14 has one LDLrA, three CUB, ten Sushi, nineteen EGF, two discoidin, one laminin G and one hyalin repeat; CTL15 has one FTP, eleven Sushi and two EFh; CTL16 has one FTP and four Sushi. While lineage-specific expansion of the gene family is remarkable in D. melanogaster and A. gambiae , we have not found any evidence for that in T. castaneum (or A. mellifera): Tc-CTL1, 2, 5, 6, 8, 9, 12 through 16 have clear orthologs in the other insect species whereas Tc-CTL7, 10 and 11 are deeply rooted (Additional data file 3).
Galectins are β-galactoside recognition proteins with significant sequence similarity in their carbohydrate-binding sites characteristic of the family. Drosophila DL1 binds to E. coli and Erwinia chrysanthemi . Leishmania uses a sandfly galectin as a receptor for specific binding to the insect midgut . Tc-galectin1 has two carbohydrate recognition domains; Tc-galectin2 and 3 are orthologous to Am-galectin1 and 2, respectively (Additional data file 4).
All fibrinogen-related proteins (FREPs) contain a carboxy-terminal fibrinogen-like domain associated with different amino-terminal regions. In mammals, three classes of FREPs have been identified: ficolin, tenascins, and microfibril-associated proteins . They take part in phagocytosis, wound repair, and cellular adhesion . In invertebrates, FREPs are involved in cell-cell interaction, bacterial recognition, and antimicrobial responses [34, 35, 36]. The Tribolium genome contains seven FREP genes, which fall into three groups (Additional data file 5): the expansion of group I yielded four family members: Tc-FREP1 through 4. Sitting next to each other on chromosome 3, these beetle genes encode polypeptides most similar to angiopoietin-like proteins. During angiogenesis, the human plasma proteins interact with tyrosine kinase receptors (for example, Tie) and lead to wound repair and tissue regeneration . In group II, Tc-FREP5 is orthologous to Dm-scabrous, which is required for Notch signaling during tissue differentiation . Interestingly, Notch is also needed for proper differentiation of Drosophila hemocytes . Group III includes Tc-FREP6, Tc-FREP7, Ag-FREP9 and Dm-CG9593. No major expansion has occurred in the beetle or honeybee, in sharp contrast to the situations in the fly and mosquitoes - there are 61 FREP genes in the A. gambiae genome .
Thioester-containing proteins (TEPs), initially identified in D. melanogaster , contain a sequence motif (GCGEQ) commonly found in members of the complement C3/α 2-macroglobulin superfamily. After cleavage activation, some TEPs use the metastable thioester bond between the cysteine and glutamine residues to covalently attach to pathogens and 'mark' them for clearance by phagocytosis . One of the 15 TEPs in Anopheles, Ag-TEP1, plays a key role in the host response against Plasmodium infection and ten other Ag-TEPs are results of extensive gene duplications. This kind of family expansion did not happen in the beetle (or bee): Tribolium encodes four TEPs, perhaps for different physiological purposes. Our phylogenetic analysis supports the following orthologous relationships: TcA-AmA-Ag13-Dm6, TcB-AmB-Ag15-Dm3, and TcC-AmC (Additional data file 6).
Extracellular signal transduction and modulation
Similar to the alternative and lectin pathways for activation of human complements, insect plasma factors play critical roles in pathogen detection, signal relaying/tuning, and execution mechanisms. Serine proteinases (SPs) and their noncatalytic homologs (SPHs) are actively involved in these processes. Some SPs are robust enzymes that hydrolyze dietary proteins; others are delicate and specific - they cleave a single peptide bond in the protein substrates. The latter interact among themselves and with pathogen recognition proteins to mediate local responses against nonself. The specificity of such molecular interactions could be enhanced by SPHs, adaptor proteins that lack proteolytic activity due to substitution of the catalytic triad residues. SPs and SPHs constitute one of the largest protein families in insects [29, 41, 42]. We have identified 103 SP genes and 65 SPH genes in the Tribolium genome, 77 of which encode polypeptides with a SP or SP-like domain and other structural modules. These include thirty SPs and eighteen SPHs containing one or more regulatory clip domains. Clip-domain SPs, and occasionally clip-domain SPHs, act in the final steps of arthropod SP pathways . Other recognition/regulation modules (for example, LDLrA, Sushi, CUB and CTL) also exist in long SPs (>300 residues), some of which act in the beginning steps of SP pathways.
Intracellular signal pathways and their regulation
The IMD pathway is critical for fighting certain Gram-negative bacteria in Drosophila. Upon recognition of diaminopimelate-peptidoglycan by PGRPs, the 'danger' signal is transduced into the cell through IMD (Figure 5b). IMD contains a death domain that recruits dFADD (dTAK1 activator) and Dredd (a caspase). Active dTAK1 is a protein kinase that triggers the JNK pathway (through Hep, Basket, Jra and Kay) and Relish phosphorylation (through Ird5 and Kenny). The presence of 1:1 orthologs in T. castaneum strongly suggests that IMD-mediated immunity is conserved in the beetle. Furthermore, the modulation of these pathways may also resemble each other - we have identified putative 1:1 orthologs of IAP2, Tab2 and caspar in the Tribolium genome (Figure 5b).
The transcription of Drosophila TEPs and some other immune molecules is under the control of the JAK-STAT pathway . This pathway, triggered by a cytokine-like molecule, Upd3, promotes phagocytosis and participates in an antiviral response. Based on sequence similarity, we predict that the conserved signaling pathway in the beetle is composed of the orthologs of Dm-Domeless, Hopscotch and STAT92 (Figure 5c). However, we have not identified any ortholog of Dm-upd, upd2, or upd3, possibly due to high sequence variation in the cytokine-like proteins.
Phenoloxidases are copper-containing enzymes involved in multiple steps of several immune responses against pathogens and parasites (that is, clot reinforcement, melanin formation, ROS/RNS generation, and microbe killing) . Synthesized and released as an inactive zymogen, proPO requires a SP cascade for its cleavage activation. SPHs and serpins ensure that the proteolytic activation occurs locally and transiently in response to infection. We have identified three proPO genes in the Tribolium genome, designated proPO1, 2 and 3. Tc-proPO2 and proPO3 are 98.8% identical in nucleotide sequence and 99.6% identical in amino acid sequence. In the aligned coding regions (2,052 nucleotides long), 21 of the 24 substitutions are synonymous, corresponding to 0.0102 changes/site. These two genes are 530 kb apart and their aligned intron regions are 88.5% identical. Using the relative rate of nucleotide substitutions derived from an analysis of Drosophila alcohol dehydrogenase genes , we estimate that Tc-proPO2 and Tc-proPO3 arose by gene duplication approximately 0.6 million years ago. The phylogenetic analysis suggests that such evolutionary events are sporadic for this family: the total numbers of proPO genes in different insect species did not change significantly, except for the malaria mosquito (Additional data file 8). Of the nine Ag-proPO genes, eight arose from gene expansion that occurred early in the mosquito lineage , some of which encode phenoloxidases for melanization.
Local production of free radicals is a critical component of the acute-phase oxidative defense, involving nitric oxide synthase, NADPH oxidase, peroxidase, phenoloxidase and other enzymes [53, 55]. Due to the cytotoxicity of ROS and RNS, their conversion and concentrations must be tightly regulated by superoxide dismutases (SODs), glutathione oxidases (GTXs), catalases, thioredoxins, thioredoxin reductases, melanin intermediates, and certain metal ions. Changes in the free radical levels by gene mutation or knock-down affect the fecundity and antimalarial response of the mosquito . We have annotated some of these genes in Tribolium, including peroxidases, GTXs, SODs, peroxiredoxins (TPXs) and catalases. T. castaneum GTX1-GTX2 and TPX2-TPX6 gene pairs are results of recent gene duplications, whereas several orthologous relationships have been identified in the SOD and TPX families in the phylogenetic analysis (Additional data file 9).
With the genome sequence available, we are able to use the other AMP sequences to identify homologous genes that are not specified in beetles. Cecropins were mostly identified in moths and flies - there was only one report on cecropin from a coleopteran species, Acalolepta luxuriosa . In Tribolium, we find a single close homolog of the Acalolepta cecropin, although a frame shift in a run of seven adenosines indicate that this is a pseudogene (Tc00499). Closely linked to Tc00499 on chromosome 2 are two genes that encode cecropin-related peptides of unusual structure, with proline- and tyrosine-rich carboxy-terminal extensions (Tc-cecropin2 and Tc00500). These observations indicate that cecropins may widely exist in beetles. Attacins were found only in lepidopteran and dipteran species. We have identified a cluster of three attacin genes (Tc07737-07739) on Tribolium chromosome 4. Although we failed to identify a Drosomycin homolog in the beetle, our search resulted in a low-score hit of a cysteine-rich sequence. The corresponding gene (Tc11324) encodes a 104 residue polypeptide containing 2 whey acidic protein motifs. While mammalian proteins with this motif possess antibacterial activities , expression and biochemical analyses are needed to test if the Tribolium protein has a similar function. Due to the presence of species-specific AMPs and severe sequence diversity of these molecules, our homology-based search has probably missed some AMP genes. Should there be a thorough exploration by sequence similarity, biochemical separation and activity assays (not only against Gram-positive and Gram-negative bacteria, but also against yeasts and filamentous fungi), we expect the total number of AMPs (currently 12) in T. castaneum may approach that (20) in D. melanogaster. In addition to these, we have found a cluster of four lysozyme genes in the Tribolium genome (Additional data file 10). Similar but independent family growths have occurred in different insect groups, giving rise to thirteen such genes in Drosophila, eight in Anopheles, three in Apis, and four in Tribolium.
Transcriptional regulation is not limited to pattern recognition molecules or extracellular signal mediators/modulators: we detected differential expression of ligand and their receptors (for example, Tc-spätzle1, Toll-1 through Toll-4, and IMD). mRNA level changes for the latter genes were small except for IMD (Figure 8). Toll-3 and Toll-4 induction after the C. albicans or M. luteus challenge was apparent, although not as notable as IMD. The subtle changes in Toll-1 transcript levels were somewhat different from those of Toll-2, -3 and -4, indicating that there could be functional differences and overlaps in antimicrobial responses for these closely related receptors (Figure 4).
We have also examined genes whose products are plasma proteins directly involved in microbe immobilization or killing. The transcripts of Tc-proPOs, lysozyme1 or lysozyme4 did not significantly change when compared with the controls, whereas those of Tc-lysozyme2 and 3 increased remarkably (Figure 8). The most dramatic increase in mRNA levels occurred in the AMP group of effector molecules, including Tc-attacin2, cecropin3, coleoptericin1, defensin1, and defensin2.
Cluster analysis of the expression patterns has revealed several trends of the transcriptional control of these immune genes. Buffer injected and uninjured adults form one cluster with the lowest mRNA levels, whereas E. coli- and S. cerevisiae-treated insects have the next higher level of overall gene expression (Figure 8). The yeast-injected beetles, instead of grouping with E. coli-treated insects, are found in the same cluster with C. albicans-challenged adults. Interestingly, immune responses toward the opportunistic fungal pathogen are greater than those toward S. cerevisiae, an environmental non-pathogen present in the diet. The responses toward M. luteus and C. albicans were significantly stronger than those towards E. coli, implying that the Toll pathway triggered by the Gram-positive bacteria and filamentous fungi more effectively up-regulated target gene expression than the IMD pathway did, which may be activated by the Gram-negative bacterial infection (Figure 5).
Through this comparative genome analysis, we have provided evidence in the red flour beetle for the functional conservation of intracellular immune signaling pathways (Toll, IMD and JAK/SAT) and for the evolutionary diversification of over 20 families of proteins (for example, PGRPs, clip-domain proteins, serpins, Toll-related receptors, antimicrobial proteins and scavenger receptors) involved in different mechanisms of insect defense against infection. The observed differences in conservation are likely related to distinct needs for specific molecular interactions and changes in microorganisms encountered by the host insects. For instance, Drosophila Myd88, Tube, Pelle, Pellino and TRAF, which form a macromolecular complex with the Toll/interleukin 1 receptor domain (Figure 5), have 1:1 orthologs in Anopheles, Apis and Tribolium. In contrast, family expansion and sequence divergence in the PGRP and AMP families are perhaps important for specific recognition and effective elimination of evolving pathogens.
The summary of putative immune gene counts, families and functions (Additional data file 11) suggests that T. castaneum has a more general defense than A. gambiae does. While this system is critical for the survival of this beetle, we are unclear whether or not it correlates with the prosperity of coleopteran insects. Drastic lineage-specific expansions seem sporadic and, in most cases, Tribolium paralog counts are lower than those of Anopheles or Drosophila (but are considerably higher than of Apis). The only exceptions are the clip-domain SP/SPH and serpin families: 48, 41 and 37 proteinase-related genes and 31, 14 and 28 inhibitor genes are present in the beetle, mosquito and flies, respectively. Because clip-domain SPs are often regulated by serpins, positive selection may have played a role in the converted evolution of both families and in the maintenance of homeostasis.
This comparative analysis has also uncovered interesting genes and gene families for future research. For instance, the existence of a 1:1 ortholog of Drosophila PGRP-LE in Tribolium (but not in Anopheles or Apis) may allow us to test whether or not TcPGRP-LE has a similar function. It can be interesting to explore the molecular mechanisms and evolutionary pathways of the large serpin and SP gene clusters in the beetle. The presence of TcToll-1 through -4 and subtle changes in their mRNA levels after immune challenges call for detailed analysis of their transcriptional regulation and physiological functions. Of course, the proposed extracellular and intracellular signaling pathways need to be tested, even though we have confidence in their general structures. The possible AMP function of Tc11324, which contains two whey acidic protein motifs, needs to be established experimentally.
It is noteworthy that the functions of Tribolium immunity-related genes are mostly assumed based on sequence similarity to studied proteins in Drosophila or other insect species. Functional analyses using the strong reverse genetic techniques available in Tribolium are necessary to test the hypotheses. Nevertheless, the framework of information established in this work should help clarify immune functions in an important agricultural pest from the most diverse insect order and a species that can serve as a tractable model for an innate immune system more generally.
Materials and methods
Database search and sequence annotation
Known defense proteins from other insects were used as queries to perform BLASTP searches of Tcastaneum Glean Predictions (2005.10.11) . Protein sequences with E-values lower than 0.1 were listed, and every 5th sequence was retrieved for use as a query for another round of search. Based on the combined lists, respective protein sequences were retrieved, compiled in the order of ascending E-values, and improved by two methods. Firstly, Tcastaneum ESTs (2005.9.20) at the same HGSC site were searched with the corresponding nucleotide sequences to identify possible cDNA clones. The EST sequences were assembled using CAP3  and the resulting contigs were used in pairwise comparison  to validate the gene predictions. Secondly, retrieved protein sequences were analyzed by CDART , PROSITE , and SMART  to detect conserved domain structures required for specific functions. Necessary changes were made after each step to improve the original predictions. Chromosomal location and exon-intron boundaries for each annotated sequence were acquired from Genboree . To locate orthologs not identified by BLASTP, Tribolium Genome Assembly 2.0  was searched using TBLASTN. The hits detected were analyzed using multiple gene prediction tools Genescan and Genemark [71, 72]. All curated sequences then were deposited in the annotation database  as a part of Tribolium Genome Assembly 2.0.
Unless otherwise specified, full-length Tribolium sequences were aligned with their homologs from other insects, including D. melanogaster, A. gambiae and A. mellifera. The sequences were retrieved from NCBI , Flybase , or Ensembl . Multiple sequence alignments were carried out using ClustalX  and Blosum series of weight matrices . Phylogenetic trees were constructed based on algorithm of neighbor-joining using PHYLIP  or maximum-parsimony using PAUP . The divergence time of Tc-proPO2 and proPO3 were calculated using the rate of 1.7 × 10-8 synonymous substitutions/nucleotide/year derived from the Drosophila species .
Gene expression analysis
To study pathogen-induced gene expression, adult red flour beetles (approximately 240 per group) were pricked at the ventral thorax with needles dipped in sterile phosphate-buffered saline or the buffer containing concentrated live E. coli, M. luteus, C. albicans or S. cerevisiae cells. Uninjured and aseptically injured insects were employed as controls. Total RNA samples were extracted from the control and challenged insects (approximately 160 per group) 24 h later, using Micro-to-mid RNA Purification System (Invitrogen, Carlsbad, CA, USA). After DNA removal, each RNA sample (1.0-3.4 μg), oligo(dT) (0.5 μg, 1 μl) and dNTPs (10 mM each, 1 μl) were mixed with diethyl pyrocarbonate-treated H2O in a final volume of 12 μl, and denatured at 65°C for 5 minutes. First strand cDNA was synthesized for 50 minutes at 42°C using SuperScript Reverse Transcriptase (200 U/μl, 1 μl; Invitrogen) mixed with 5 × buffer (4 μl), 0.1 M dithiothreitol (2 μl), RNase OUT (40 U/μl, 1 μl; Invitrogen) and the denatured RNA sample (12 μl). Specific primer pairs were designed for a total of 35 immunity-related genes (Additional data file 12) using Primer 3  with annealing temperatures of 59.5-60.5°C and expected product sizes of 80-150 bp. Each primer pair was located in adjacent exons flanking an intron. Real-time PCR was performed in parallel reactions on 96-well microtiter plates using Taq DNA polymerase (1 U; Roche Applied Sciences, Indianapolis, IN, USA), 1 × buffer, 1 mM dNTP mix, 2 mM MgCl2, 0.2 μM primers, 1 × SYBR-Green I dye (Applied Biosystems, Foster City, CA, USA) and 10 nM fluorescein. Amplifications were enacted on an iCycler thermal cycler (Bio-Rad, Hercules, CA, USA) with a profile of 95°C for 5 minutes followed by 40 cycles of 94°C for 20 s, 60°C for 30 s, 72°C for 60 s and 78°C for 20 s . SYBR green fluorescence was measured during the 78°C step in each cycle and the cycle numbers for each target and control gene were recorded when the fluorescence passed a predetermined threshold. Proper dissociation and correct size of the products were examined by melting curve analysis and agarose gel electrophoresis, respectively. The real-time PCR was repeated twice and, in each of the three experimental replicates, the transcripts were normalized relative to the levels of Tribolium ribosomal protein S3. Averaged transcript abundance values (Ctcontrol - Cttarget) were then compared across genes and samples using average-linking clustering (Cluster 3.0) and visualized using TreeView .
Additional data files
The following additional data are available with the online version of this paper. Additional data file 1 is a table listing immunity-related genes in T. castaneum. Additional data file 2 is a figure showing sequence alignments of βGRPs and GNBPs. Additional data file 3 is a figure showing sequence alignments of CTLs. Additional data file 4 is a figure showing sequence alignments of galectins. Additional data file 5 is a figure showing sequence alignments of FREPs. Additional data file 6 is a figure showing sequence alignments of TEPs. Additional data file 7 is a figure showing sequence alignments of Spätzle-related proteins. Additional data file 8 is a figure showing sequence alignments of proPOs. Additional data file 9 is a figure showing sequences of GTX, SOD and TPX. Additional data file 10 is a figure showing sequence alignments of lysozymes. Additional data file 11 is a table listing functions, families, and counts of putative defense proteins from D. melanogaster, A. gambiae, A. mellifera and T. castaneum. Additional data file 12 is a table listing oligonucleotide primers used in expression analysis by real-time PCR.
We greatly appreciate our colleagues in the Tribolium genome sequence consortium for the gene prediction. Dr Thomas Phillips kindly provided the insects for immune challenges and RT-PCR experiments. We would also like to thank Drs Ulrich Melcher, Jack Dillwith, and Maureen Gorman for their helpful comments on the manuscript. This work was supported by the National Institutes of Health Grants GM58634 (to HJ). The article was approved for publication by the Director of Oklahoma Agricultural Experimental Station and supported in part under project OKLO2450.
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