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Tissue-Specific Transcriptomics of the Exotic Invasive Insect Pest Emerald Ash Borer (Agrilus planipennis)

Background The insect midgut and fat body represent major tissue interfaces that deal with several important physiological functions including digestion, detoxification and immune response. The emerald ash borer (Agrilus planipennis), is an exotic invasive insect pest that has killed millions of ash trees (Fraxinus spp.) primarily in the Midwestern United States and Ontario, Canada. However, despite its high impact status little knowledge exists for A. planipennis at the molecular level. Methodology and Principal Findings Newer-generation Roche-454 pyrosequencing was used to obtain 126,185 reads for the midgut and 240,848 reads for the fat body, which were assembled into 25,173 and 37,661 high quality expressed sequence tags (ESTs) for the midgut and the fat body of A. planipennis larvae, respectively. Among these ESTs, 36% of the midgut and 38% of the fat body sequences showed similarity to proteins in the GenBank nr database. A high number of the midgut sequences contained chitin-binding peritrophin (248)and trypsin (98) domains; while the fat body sequences showed high occurrence of cytochrome P450s (85) and protein kinase (123) domains. Further, the midgut transcriptome of A. planipennis revealed putative microbial transcripts encoding for cell-wall degrading enzymes such as polygalacturonases and endoglucanases. A significant number of SNPs (137 in midgut and 347 in fat body) and microsatellite loci (317 in midgut and 571 in fat body) were predicted in the A. planipennis transcripts. An initial assessment of cytochrome P450s belonging to various CYP clades revealed distinct expression patterns at the tissue level. Conclusions and Significance To our knowledge this study is one of the first to illuminate tissue-specific gene expression in an invasive insect of high ecological and economic consequence. These findings will lay the foundation for future gene expression and functional studies in A. planipennis.


Introduction
The insect midgut and fat body are important metabolic tissues and represent key physiological interfaces during interactions with hosts. While both tissues are highly specialized and play major roles in the life of insects, they possess discrete physiological functions essential for growth and development. The midgut plays important roles in digestion, antioxidant defense and is thought to be one of the primary interfaces in countering the effects of dietary toxins [1][2][3][4][5], xenobiotics (ex. insecticides) and potential pathogens [6]. On the other hand, the insect fat body is a dynamic tissue, which plays vital roles in intermediary metabolism, energy storage/utilization, detoxification and immune response [7,8]. Remarkably, functions performed by the insect fat body are correlated to those of adipose tissue and liver of vertebrates. The combined action of the factors associated with the midgut and fat body could determine the capacity of an insect to adapt to various ecological niches and thereby evolve.
Beetles are the most predominant group of insects constituting about 25% of all known life-forms [9]. Within Coleoptera, the Buprestidae, known commonly as metallic wood-borers, remains one of the most understudied groups of insects despite their ecological importance in forests and the status of some species as key pests of trees. For example, emerald ash borer (Agrilus planipennis Fairmaire) which is endemic to Asia, has emerged as a devastating pest of ash trees in North America where it continues to kill millions of ash trees, primarily in the Midwestern United States and Ontario, Canada [10,11]. All North American ash that it has encountered, including green ash (Fraxinus pennsylvanica Marshall), white ash (F. americana L.) and black ash (F. nigra Marshall) are highly susceptible [11][12][13]. If A. planipennis sustains its current rate of invasion, it is considered to have the potential to decimate ash on a continental scale [14,15] with economic and ecological impacts reminiscent of the invasions of the chestnut blight and Dutch elm disease pathogens [16,17].
Larvae of A. planipennis feed on the phloem and cambial tissues of ash trees, which usually results in the formation of S-shaped galleries that disrupt nutrient and water translocation, generally killing the trees in 3-5 years following attack [10]. A. planipennis is endemic to Far East Asia, including China, Korea, Japan and Siberia [18]. However, reports indicate that A. planipennis is rarely considered a pest in Asia [19,20], where Manchurian ash (F. mandshurica Rupr) is a primary host [21,22], and infestations appear restricted to stressed trees [23]. This implies that Manchurian ash may be generally resistant, with weakened trees preferentially colonized. A common garden experiment in Michigan confirmed that Manchurian ash has a much higher level of resistance to A. planipennis than do common North American ash species [24] and it has been reported that the resistance may be mediated by phenolic compounds in the phloem such as hydroxycoumarins and phenylethanoids, among other possible mechanisms [25].
Despite its status as a key pest, no functional genomics data exist for A. planipennis. In general, little is known at the tissue-level about the physiology-driven molecular strategies that invasive insects deploy to adapt to their new environment. This lack of fundamental molecular knowledge hinders our understanding of the insect's biology. However, the advent of second-generation sequencing such as 454 pyrosequencing [26] and Illumina/Solexa [27] offers a unique opportunity to study functional genomics [28] in non-model organisms. For example, the 454 pyrosequencing strategy has been successfully applied to unravel transcriptomic signatures in the tobacco hornworm, Manduca sexta L. [3], the soybean aphid, Aphis glycines [29] and the poplar leaf beetle, Chrysomela tremulae F. [30]. Here, we present data derived from 454 pyrosequencing of the midgut and fat body of A. planipennis larvae. Results obtained from this study will provide the basis for performing future gene expression and functional studies in A.
planipennis. Furthermore, the data will improve our understanding of the biology of this key invasive pest at the molecular level.

Transcriptome analysis
We obtained 126,185 transcriptomic reads for the midgut and 240,848 reads for the fat body of third-instar A. planipennis larvae. The reads for both tissues were assembled using Newbler program (Roche) after the removal of adapter sequences. To achieve better consistency, the contigs and singletons of the transcripts were renamed in the format ''EABMG000001'' with ''EAB'' for emerald ash borer, ''MG'' for midgut, and ''000001'' for an arbitrarily assigned number. Similar nomenclature was adopted to describe the fat body transcriptomic sequences with MG replaced by FB for fat body.
After the assembly of the sequences, 25,173 high quality ESTs (2,218 contigs and 23,495 singletons) were obtained for the midgut and 37,661 ESTs (5,376 contigs and 32,285 singletons) for the fat body ( Table 1). The singleton sequences for the midgut ranged from 50 bp to 710 bp with an average length of 259 bp and total length of 6,091,300 bp; while the contig sequences ranged from 36 bp to 3,395 bp with an average length of 688 bp and total length of 1,526,781 bp. The singleton sequences for the fat body ranged from 50 bp to 606 bp with an average length of 314; while the contiguous sequences ranged from 38 bp to 4,501 bp with an average length of 825 bp and total length of 4,433,073 bp ( Figure 1).
Among the midgut transcripts, 9,066 (36%) showed significant similarity (E value , 1e2 5 ) to proteins in the GenBank nr database, while 14,253 (38%) sequences of the fat body ESTs showed significant hits ( Table 1). The majority of the transcripts with similar sequences in the database (95%) matched to arthropodal proteins. The remaining midgut and fat body transcripts were similar to proteins of non-insect eukaryotes (5%) and bacteria (0.02%). A total of 14 sequences were similar to viral proteins and artificial sequences; and one midgut sequence matched to an Archaea protein (Table 1).  Table S1).

Gene Ontology
The obtained unigenes were assigned Gene Ontology (GO) based on significant homologies to proteins in GenBank. Since the two EST databases represent two different tissues, GO studies were performed separately to allow tissue-specific comparisons. GO terms were assigned to 7,304 midgut transcriptomic sequences (2,527 Biological Process, 585 Cellular Component and 1,169 Molecular Function; Figure 3 and Supplemental Table S2) and to 10,074 fat body transcriptomic sequences (2,827 Biological Process, 646 Cellular Component and 1,454 Molecular Function; Figure 4 and Supplemental Table S2).

Metabolic pathways
We identified 115 KEGG metabolic pathways for the midgut and 122 for the fat body (Supplemental Table S3 and S4). Of the pathways identified, it was interesting to note that the beta-lactam resistance pathway was midgut-specific. However, at this point it is unclear as to what the functional role of this pathway may have specifically in the midgut. The specific pathways identified in the fat body were related to steroid biosynthesis, tetracycline biosynthesis, D-arginine and D-ornithine metabolism, ubiquinone and other terpenoid backbone biosynthesis. Besides these pathways, both the midgut and fat body databases also revealed transcripts for biosynthesis of alkaloids (derived from ornithine), lysine, nicotinic acid, histidine and purine.

Protein domains
A Pfam domain search yielded 5,876 domains in 4,925 midgut transcripts and 8,852 domains in 7,205 fat body transcripts ( Table 2). Among the identified Pfam domains, chitin binding Peritrophin-A (CBP-A) was found to be the highest in occurrence  with 248 transcripts (4.2%) in the midgut and a mere 16 sequences (0.2%) in fat body. CBP-A domain is commonly found in proteins that constitute the peritrophic matrix (PM), a semipermA. planipennisle membrane that lines the gut of most insects [6,32,33]. PM assists in digestion and protecting the midgut epithelium from abrasion as well as from pathogens including bacteria, viruses and parasites [34,35]. The presence of 0.2% fat body sequences with CBP-A domains is probably due to contamination during dissections and sample collection. Several domains pertaining to detoxification enzymes were also identified in both the midgut and fat body transcripts of A. planipennis. First, compared to the fat body, the midgut showed a greater number of sequences matching domains of carboxylesterases (CEs). A similar high frequency of CEs was reported in the midgut of C. tremulae [30]. CEs are general detoxification enzymes involved in the resistance to carbamates, pyrethroids and organophosphate insecticides [36]. However, it is not clear what their role may be in the detoxification of plant allelochemicals [37]. Second, cytochrome P450 domains were found more frequently in fat body sequences than those of the midgut. These enzymes have been reported to detoxify a wide-range of xenobiotics including plant toxins and synthetic chemicals in an array of insect species [38].
WD-40 repeats were found in both midgut and fat body sequences. WD-repeat protein and its subunit domains were found significantly higher in fat body compared to the midgut ( Table 2). WD-repeat proteins play an important role in RNA processing, signal transduction, cytoskeleton assembly, cell division and proteinprotein interactions [29,39]. In the fat body we identified the katanin domain, which is part of a microtubule severing protein (heterodimer of 60 and 80 kDa subunits) that targets the centrosome using WD-40 containing subunit [40]. Other identified domains that are potentially involved in signal transduction and cytoskeleton related functions were GTPase Ras families [41]. P-element wimpy testis-induced (PIWI) -interacting RNAs (piRNA) play an important role in gene regulation in addition to silencing retrotransposons and repetitive sequences during germline development [42,43]. We identified some of the domains of proteins that are involved in the piRNA synthesis pathway. Interestingly, these domains were almost restricted to the fat body sequences of A. planipennis and included PIWI-like proteins and Tudor domain that are essential for piRNA production [42,44]. The midgut sequences revealed only Tudor domains. piRNA differ from micro-RNAs (miRNAs) and small interfering RNAs (siRNAs) in several ways: 1) they interact only with PIWI and not with Argonaute; 2) piRNAs range from 24 to 31 nucleotide (nt) length compared to siRNAs and miRNAs, which are typically 21nt; and 3) piRNAs may positively regulate mRNA stability and translation whereas miRNAs and siRNAs have negative effect on transcriptional and translational process [45,46]. To-date, the function of piRNA is unclear and deciphering the functional role of molecules involved in piRNA synthesis would contribute novel insights into gene regulation in insects.
Apart from the above-described domains we found a significant number of other domains pertaining to trypsins, glycosyl hydrolase family, sugar transporters, protein kinase domain, mitochondrial protein domains, C 2 H 2 type zinc finger domains and RNA recognition motifs (Supplemental Table S5). The presence of a high number of trypsin and trypsin-like domains suggest that the gut of A. planipennis is predominantly composed of serine-proteases, which seems to be also one of the major classes of digestive proteases in the midgut of the beetle C. tremulae [30].

Genes of interest
We are primarily interested in elucidating the functions of the genes involved in detoxification/antioxidant response (Table 3). Of particular interest are cytochrome P450s, which are known to be one of the most rapidly radiating groups of detoxifying enzymes in insects [47]. Cytochrome P450s metabolize toxic substrates including allelochemicals and insecticides to less harmful excretable forms via hydroxylation/oxidation [47,48]. Cytochrome P450s have been classified into four large clades: CYP2, CYP3, CYP4, and Mitochondrial clade [47]. In this study, the predominant occurrence of A. planipennis cytochrome P450s was members of the CYP3 clade, which is in agreement with those reported in various insect orders including Diptera, Lepidoptera and Coleoptera [47]. The genome of the red flour beetle T. castaneum encodes about 147 cytochrome P450s of which 70 belong to the CYP3 clade [31]. To begin ascertaining putative function, we assessed the mRNA levels of candidate A. planipennis cytochrome P450 genes belonging to each of the four clades in different larval tissues (midgut, fat body and cuticle).
Members of the CYP3 clade (mainly CYP6 and CYP9) revealed distinct expression patterns. Of the two A. planipennis CYP6 genes assayed, EABMG023904 showed highest mRNA levels in the midgut while contig EABMG001813 showed higher transcript level in the fat body ( Figure 5A). Similar mRNA levels of tissue-specific CYP6 genes have been observed in other insect species [49][50][51]. It is thought that the high expression levels of CYP6 genes were associated with metabolism of plant allelochemicals and insecticides [52]. However, qPCR analysis for a CYP9 member revealed highest mRNA levels in larval cuticle of A. planipennis ( Figure 5A). CYP9 in A. planipennis larvae could be involved in xenobiotics metabolism as reported in other insect taxa [52,53].
Although the CYP4 clade consists of numerous diverse genes, it seems to be the least studied group of cytochrome P450s in insects [48]. In this study, larval A. planipennis midgut showed the highest mRNA level for one of the CYP4 assayed ( Figure 5B). Transcript levels in the fat body were significantly higher compared to levels in cuticle but significantly lower than the levels for the midgut. These observations are in agreement with CYP4 mode of action on dietary plant toxins reported in Lepidoptera (e.g. Manduca sexta; [54]) as well as against insecticides in Coleoptera (e.g. Diabrotica virgifera virgifera Leconte [55]). Apart from xenobiotic metabolism, members of CYP4 are also reported to have putative roles in ecdysteroid metabolism [56].
Of the CYP2 clade homologs identified in A. planipennis midgut, we assessed the expression of CYP306, which belongs to a set of cytochrome P450s called Halloween genes or otherwise known as the phantom gene, phm [57]. These genes are thought to be involved in ecdysone metabolism and therefore are important for insect growth and development. The CYP306 homolog of A. planipennis showed highest mRNA levels in the midgut and cuticle ( Figure 5C). Similar tissue-specific profiles of CYP306 (phm) were observed in larva of M. sexta [57].
Mitochondrial P450s are very specific to animals and are not found in fungi and plants [48]. Insects have two types of mitochondrial P450, a conserved group involved in physiological functions and a diverse group known to participate in the detoxification of xenobiotics. Expression analysis of an A. planipennis CYP12 gene belonging to the mitochondrial clade revealed highest mRNA levels in the midgut compared to cuticle and fat body ( Figure 5D), similarly to a study on resistance of D. melanogaster larvae to lufenuron, which was attributed to high expression of a CYP12 in the midgut and Malphigian tubules [58].
The above results suggest a potential involvement of cytochrome P450s in metabolism of host allelochemicals by A. planipennis larvae and perhaps in other physiological functions such as hormonal biosynthesis. However, additional experiments at the protein and transcript level (RNA interference) that elucidate gene function of these cytochrome P450s could provide insights into alternate control targets as in other insect systems [51].

Molecular markers
We predicted 317 simple sequence repeats (SSRs or microsatellites) in A. planipennis midgut sequences and 571 SSRs in fat body sequences ( Table 4). The majority of SSRs were either di-nucleotide (58 for midgut and 94 for fat body) or trinucleotide (223 for midgut and 428 for fat body) repeats; while 26 and 46 sequences of the midgut and fat body, respectively, were predicted to be tetranucleotide repeats and 10 and 3 sequences of the midgut and fat body, respectively, were predicted to be penta-nucleotide repeats. The characteristics of these SSRs including length, start, stop motif units and foot print are summarized in Supplemental Table S6. Interestingly, we also identified 137 putative single nucleotide polymorphisms (SNPs) in 45 sequences of the midgut and 347 SNPs in 143 sequences of the fat body (Table 5) (Supplemental Table S7). The exploitation of markers which are linked to coding regions would facilitate the selection of polymorphic markers for A. planipennis [59]. However, prior to making any inferences of the predicted molecular markers in A. planipennis, validation has to be performed to account for false positives.

Midgut microbiota
A small subset of the sequences (79 of 14680) showed closer similarity to microbial, rather than animal genes. They might thus represent low levels of microbial contamination in the dissection, or even horizontal transmission of genes from symbiotic microbes to the insect host (Supplemental Table S8). First, putative endoglucanases were found that matched to Streptomyces species. Similar genera of microbiota in A. planipennis were identified by Vasanthakumar et al. [60]. In particular, they isolated Streptomyces sp. from midgut of larva; Erwinia sp. and Burkholderia cepacia from the guts of adult beetles. Second, putative endo-polygalacturonases were identified in the midgut of A. planipennis. Given that the diet of larval A. planipennis is primarily phloem-based, the recovered endoglucanases and endo-polygalacturonases could facilitate in breakdown of plant cell walls [61] resulting in the release of nutrients required for larval growth and development.
A single bacterial sequence in the midgut transcriptome showed similarity with a Wolbachia sequence, an ubiquitous endosymbiont found across various insect taxa and nematodes [62][63][64][65]. Wolbachia is thought to interfere with insect reproduction through a process known as feminization, in which the sex ratios within a population become female-skewed [66,67].

Conclusions
This study represents the first report to elucidate tissue-specific gene expression in A. planipennis. Our analysis of A. planipennis midgut and fat body has revealed commonalities as well as potentially significant differences with other insect model systems and is beginning to provide insights into some of the fundamental physiological processes occurring in these tissues. Some noteworthy results of this study are 1) a significant number of cytochrome P450s that showed distinct tissue expression patterns; 2) a number of SNPs and microsatellite markers were predicted within the midgut and fat body databases, which upon validation could provide insights at functional polymorphism within and between tissues; and 3) some of the predicted microbial sequences encoding for enzymes involved in carbohydrate digestion and metabolism in the larval midgut of A. planipennis suggest the presence of putative endosymbionts. These traits along with the recovered sequences  involved in piRNA synthesis provide substantial insights into the physiology-driven molecular mechanisms of A. planipennis.

Insect samples
A. planipennis larvae were collected from wood-lots consisting of green ash trees in Ottawa Lake, MI and Toledo, OH. Larvae were extracted by stripping off the bark of infested ash trees that were 10-25 cm in diameter at breast height. The ash trees sampled were alive but showed obvious symptoms of decline in response to A. planipennis colonization. The collected larvae were categorized according larval instars.

Larval dissections and RNA isolation
About 50 late 3 rd -instar larvae were dissected to extract midgut and fat body tissues. Dissections were performed as described by Vasanthakumar et al. [60]. The midgut tissue was dissected free of its contents and carefully rinsed in fresh PBS buffer prior to RNA isolation. Isolated larval midgut, cuticle and fatbody were placed in pre-chilled Trizol reagent in separate 1.5 ml eppendorf tubes. Following dissection, the tissues were stored at 280uC until RNA was isolated. The dissected tissues were homogenized separately in TRIzolH and total RNA was isolated following the manufacturer's protocol and stored at 280uC until use.

cDNA library construction
The midgut and fat body RNA samples (10-20 mg) described above were used for constructing cDNA libraries. Prior to cDNA library construction, the total RNA was subjected to quality control using the Nanodrop 2000c (Thermo Scientific) and the RNA 6000 nanochip (Agilent Technologies) at the Purdue Genomics Core Facility (West Lafayette, IN). A SMART cDNA library construction kit (Clontech) was used to construct both libraries with modifications to suit the 454 GS FLX pyrosequencing. Instead of using the recommended MMLV reverse transcriptase, Super script II reverse transcriptase from Invitrogen was used for synthesizing the firststrand cDNA, and a modified PAGE-purified CDS II primer (59 -TAG AGG CCG AGG CGG CCG ACA TGT TTT GTT TTT TTT TCT TTT TTT TTT VN -39) was used instead of the supplied one. The constructed cDNA libraries were shipped to the Purdue Genomics Core Facility for sequencing.

pyrosequencing
DNA bands of 500-800 bp were excised from the gel and purified. The isolated DNA was blunt ended, ligated to adapters and immobilized on Library Immobilization Beads. Blunt-end fragments were made with T4 polynucleotide kinase (PNK) and T4 DNA polymerase in a reaction that was held for 15 minutes at 12uC followed by 15 additional minutes at 25uC. The adaptors (Titanium A & B) used were provided with the 454 general library kit. The ligation reaction included ligase buffer, adaptors, and a ligase enzyme, which was incubated for 15 minutes at 25uC on a thermocycler. After the gaps were repaired, a single-stranded DNA library was isolated from the beads and quality controlled for the correct size using a LabChip 7500 machine. The concentration and the proper ligation of the adapters were examined using qPCR. One-quarter of a pico-titer plate was sequenced for each tissue sample (midgut and fat body) following manufacturer's protocol. The pyrosequencing was performed as per the Genome Sequencer FLX Operation (Roche).

Bioinformatic data analysis
The transcriptome sequences of A. planipennis midgut and fat body were annotated by searching against the GenBank nonredundant database using BLASTx algorithms [68] The A. planipennis transcriptome sequences were compared to proteins of the fruit fly Drosophila melanogaster, the African malaria mosquito Anopheles gambiae, and the red flour beetle Tribolium casteneum using BLASTx algorithm [68]. Domains within the sequences were identified by searching against the Pfam database [69] using the HMMER v3 program [70]. We used Blast2GO [71,72] to predict the functions of the sequences, assign Gene Ontology terms, and predict metabolic pathways in the KEGG database [73,74]. Microsatellites/SSR markers were identified using Msatfinder version 2.0.9 program [75] and single nucleotide polymorphisms (SNPs) were predicted using the gsMapper program (Roche) with an arbitrary criterion of at least 4 reads supporting the consensus or variant.

Quantitative real-time PCR
The concentration of total RNA samples was measured using NanoDrop 2000c Spectrophotometer from Thermo Scientific. The first strand cDNA was synthesized using Superscript IITM First Strand Synthesis kit for qRT-PCR from Invitrogen, following the manufacturer's protocol. For cDNA synthesis, 1 ml of oligo (dT) [12][13][14][15][16][17][18] primer (500 mg/ml) and 1 ml of dNTPs mix (10 mM each) were added to 8 ml of total RNA sample (,0.25 mg/ml). The samples were heated in a thermo cycler at 65uC for 4 min. Then, the following reagents were added on ice to each sample: 2 ml of 106RT buffer, 4 ml of 25 mM MgCl 2 , 2 ml of 0.1 M DTT, 1 ml RNAse Out, 1 ml of SuperScript II reverse transcriptase and 1 ml of RNase H. The cycling parameters were 42uC for 90 min., 70uC for 15 min. and 37uC for 20 min. After cDNA synthesis, the concentrations of the first-strand samples were brought to 20 ng/ml, which were used as the template in qRT-PCR reactions. Cycling parameters included 95uC for 3 min and then, 40 cycles of 95uC for 10 s and 60uC for 30 s. Quantification of candidate gene expression in tissues (midgut, fat body and cuticle) was based on the Relative Standard Curve method [2] using the threshold cycle (Ct) values. For all qRT-PCR analyses the mRNA coding for a ribosomal protein (RP7) was assessed and included as the internal control. Assessment of the mRNA encoding for ribosomal proteins has been demonstrated to be an appropriate internal control in other studies [76].

Statistical analysis
Relative expression value (REV) for each gene was determined by dividing the quantities for the target samples by the quantities obtained for ribosomal protein. The REVs obtained for each gene in the tissues was analyzed by Analysis of Variance (ANOVA) (p = 0.05) using the PROC MIXED procedure of SAS (SAS Institute Inc. SAS.STAT User's Guide, Version 9.1). Two biological replicates nested with two technical replicates were included for the entire analysis. Biological replicates were included as a random effect in the model. Relative fold changes in the tissues were calculated by taking the sample (REV) that showed the least level of expression as 1X sample (calibrator,) [77]. The standard error represented the variance in the two biological replicates (2 technical replicates within each) included for this analysis.

Data Deposition
The Roche 454 reads of A. planipennis midgut and fat body were submitted to NCBI Sequence Read Archive under the accession number of SRA012309.3.