Transcriptome of the Female Synganglion of the Black-Legged Tick Ixodes scapularis (Acari: Ixodidae) with Comparison between Illumina and 454 Systems

Noble Egekwu; Daniel E Sonenshine; Brooke W Bissinger; R Michael Roe

doi:10.1371/journal.pone.0102667

. 2014 Jul 30;9(7):e102667. doi: 10.1371/journal.pone.0102667

Transcriptome of the Female Synganglion of the Black-Legged Tick Ixodes scapularis (Acari: Ixodidae) with Comparison between Illumina and 454 Systems

Noble Egekwu ¹, Daniel E Sonenshine ^1,^*, Brooke W Bissinger ², R Michael Roe ³

Editor: Erik C Johnson⁴

PMCID: PMC4116169 PMID: 25075967

Abstract

Illumina and 454 pyrosequencing were used to characterize genes from the synganglion of female Ixodes scapularis. GO term searching success for biological processes was similar for samples sequenced by both methods. However, for molecular processes, it was more successful for the Illumina samples than for 454 samples. Functional assignments of transcripts predicting neuropeptides, neuropeptide receptors, neurotransmitter receptors and other genes of interest was done, supported by strong e-values (<−6), and high consensus sequence alignments. Transcripts predicting 15 putative neuropeptide prepropeptides ((allatostatin, allatotropin, bursicon α, corticotropin releasing factor (CRF), CRF-binding protein, eclosion hormone, FMRFamide, glycoprotein A, insulin-like peptide, ion transport peptide, myoinhibitory peptide, inotocin ( = neurophysin-oxytocin), Neuropeptide F, sulfakinin and SIFamide)) and transcripts predicting receptors for 14 neuropeptides (allatostatin, calcitonin, cardioacceleratory peptide, corazonin, CRF, eclosion hormone, gonadotropin-releasing hormone/AKH-like, insulin-like peptide, neuropeptide F, proctolin, pyrokinin, SIFamide, sulfakinin and tachykinin) are reported. Similar to Dermacentor variabilis, we found transcripts matching pro-protein convertase, essential for converting neuropeptide hormones to their mature form. Additionally, transcripts predicting 6 neurotransmitter/neuromodulator receptors (acetylcholine, GABA, dopamine, glutamate, octopamine and serotonin) and 3 neurotransmitter transporters (GABA transporter, noradrenalin-norepinephrine transporter and Na+-neurotransmitter/symporter) are described. Further, we found transcripts predicting genes for pheromone odorant receptor, gustatory receptor, novel GPCR messages, ecdysone nuclear receptor, JH esterase binding protein, steroidogenic activating protein, chitin synthase, chitinase, and other genes of interest. Also found were transcripts predicting genes for spermatogenesis-associated protein, major sperm protein, spermidine oxidase and spermidine synthase, genes not normally expressed in the female CNS of other invertebrates. The diversity of messages predicting important genes identified in this study offers a valuable resource useful for understanding how the tick synganglion regulates important physiological functions.

Introduction

Ticks are obligate blood-feeding ectoparasites that serve as vectors of the causative agents of many important diseases affecting humans and animals, e.g., Lyme disease, Rocky Mountain spotted fever, tick-borne encephalitis, anaplasmosis, babesiosis and many others [1], [2]. The black-legged tick, Ixodes scapularis, is one of the most important vectors of infectious diseases to humans and animals throughout large areas of the United States and Canada [1], [3], [4]. I. scapularis is the primary vector of the microbial agents of Lyme disease, human granulocytic anaplasmosis, human babesiosis, relapsing fever and an encephalitis-causing agent (Powassan virus). Lyme disease is the most commonly reported vector-borne disease in the northern temperate zone regions of the northern hemisphere [5], [6] with 24,364 confirmed cases in the United States in 2011 [7]. Despite the many zoonotic diseases caused by tick-borne pathogens, control of ticks is still largely dependent on the use of chemical acaricides, with traditional acaricide targets being primarily neurologically-based [8]. The central nervous system (CNS) in ticks is composed of a single mass called the synganglion [9]. Despite the fact that most acaricides target the nervous system, relatively little is known about tick neurobiology and how blood-feeding and mating affect gene expression in the synganglion. Acaricide resistance has become widespread around the world causing a need for new acaricide targets or alternative methods of tick control [10]. Continued dependence on traditional chemical methods is becoming increasingly problematic in the face of ever greater demand for environmental protection and fear of chemical poisoning.

To search for alternatives to chemical control, a better understanding of the neurologic processes that control basic tick physiological processes is needed. Ixodid ticks must gorge on blood, often increasing 100 fold in body size, in order to stimulate tissue development, ecdysis, mating and reproduction [9]. Precisely how the tick's nervous system regulates these biological processes is largely unknown. During feeding, some synganglion cells, especially the neurosecretory cells, increase in size and accumulate neurosecretory substances [11], [12].

Neurohormones and neurotransmitters play key roles in tick development and physiology. Work has been conducted examining their occurrence in selected tissues in ticks such as the hemocytes [13], midgut [14], ovaries [13], and salivary glands [2], [13], [15]–[20]. However, much of the work on neurotransmitters focuses on the dopaminergic system in the salivary glands (reviewed by [21]–[23]). Much less is known about the transcribed genes in the tick synganglia, largely because of the difficulty in extracting sufficient amounts of tissue. Advances in sequencing technology now allow researchers to rapidly obtain large amounts of data from a small amount of tissue. Recent work by Simo et al. [24] showed the existence of a complex neuropeptidergic network extending to different body tissues as well as within the synganglion. However, the molecular basis for understanding just how these complex neurohormone-controlled networks regulate tick physiological functions remains to be determined. To this end, a global search to identify and characterize the numerous molecules expressed in the synganglion is needed.

Transcriptomics offers an excellent tool to approach this problem [25]. Using 454 pyrosequencing, Bissinger et al. [26] generated a cDNA library of expressed genes in the synganglion of adult female American dog ticks, Dermacentor variabilis and predicted many of the neuropeptides, neuropeptide receptors, neurotransmitters, iron transport proteins, transmembrane proteins (e.g., tetraspanins), stress reduction proteins and numerous housekeeping genes. Previous studies by Donohue et al. [27] using similar methods identified 14 neuropeptides and 5 neuropeptide receptors in this same species. Transcriptome analysis of the synganglion of the brown dog tick, Rhipicephalus sanguineus, was done with cDNA library construction in phage resistant Escherichia coli. However, this method yielded a total of only 1008 ESTs sequenced, with only 603 remaining after removal of vector contamination that could be clustered into unique transcripts [20]. Neuropeptides and their receptors have also been predicted in several species of hard ticks using bioinformatics [28] and immunohistochemistry [24] and identified by MALDI-TOF/TOF mass spectrometry [29]. Despite these few studies, there is a paucity of information about the neurobiology of ticks.

Solexa/Illumina (HiSeq), 454 pyrosequencing, and other next generation sequencing technologies provide high levels of coverage (millions of base pairs) far exceeding previous methods requiring cloning into E. coli [30]. Along with improvements in assembly technology these advances have greatly enhanced the value of transcriptomes for the study of messages encoding for diverse genes in selected tissues or whole organisms [31], even in organs as small as the tick synganglion.

Transcriptomes provide an opportunity to examine at high resolution the entire repertoire of mRNA molecules expressed in a particular organ or tissue at a particular moment in time. Transcriptomes have proven useful for the study of gene expression of many arthropods, including mosquitoes [32], bedbugs [33] and other arthropods, as well as selected organs, such as the transcriptome of the midgut of female D. variabilis [14] and the male reproductive system of this same species [34].

Here we present the first transcriptome of the synganglion of the black legged tick, I. scapularis, with identification of messages predicting neuropeptides, neuropeptide receptors, as well as receptors for neurotransmitters, oxidative stress peptides, reproduction-related peptides and many others in this important organ. We conducted BLAST matching against the published conspecific genome and gene sequence alignments which we believe are likely to increase the reliability of gene annotations in the relevant transcriptomes. Using Solexa/Illumina sequencing, we were able to create a large EST library with more than 100 million raw reads (8.49 billion bases) suitable for de novo transcriptome assembly and gene annotation/analysis. In view of the short read length (from 68–110 bp) characteristic of this technology at the time sequencing was done, we also used 454 pyrosequencing (∼450 bp read length) which was expected to offer significant advantages resulting from greater read length [25] to help recognize messages predicting genes that might have been missed by Illumina sequencing. In this report, we compare the success of these two different technologies in identifying the many messages predicting genes of interest for our understanding of synganglion function in this important tick species.

We believe that the assembled, annotated transcriptomes described in this report will provide an invaluable resource for future studies of the role of the genes involved in neurologic functioning of the tick nervous system.

Materials and Methods

Ticks

Black-legged ticks, I. scapularis were reared as previously described (Sonenshine, 1993) and originated from specimens collected near Armonk, New York, USA. Adult ticks were confined within plastic capsules attached to New Zealand white rabbits, Oryctolagus cuniculus, and allowed to feed to repletion and oviposit. Larvae and nymphs were allowed to feed on albino mice, Mus musculus. Engorged ticks were held in a Parameter Generation and Control incubator (Black Mountain, N.C.) at 26±1°C, 92±1% relative humidity and 14:10 (L:D) for oviposition or molting.

Ethics statement

All use of animals in this study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Old Dominion University Institutional Animal Care and Use Committee ((Animal Welfare Assurance Number: A3172-01). The approved protocols (#10-018 and #10-032) are on file at the Office of Research, Old Dominion University, Norfolk, Virginia. Tranquilizers (Acepromazine) were administered to the animals prior to handling to minimize anxiety and/or discomfort.

Synganglion RNA collection and sample preparation

Adult I. scapularis females, either unfed, partially-fed 5–6 days (virgin) or replete (mated), were dissected, the synganglia were excised and then washed in phosphate-buffered saline on ice (PBS: pH 7.0, 10 mM NaH₂PO₄, 14 mM Na₂HPO₄, and 150 mM NaCl). The cleaned tissues were homogenized in Qiagen RLT buffer and total RNA extracted in accordance with the manufacturer's recommendations (Qiagen, Valencia, CA). Samples were collected and frozen (−80°C) until needed.

Three different RNA samples were prepared, 1) 5.1 µg of total RNA from a mixture of ∼50 unfed, part-fed virgin and replete females for Illumina sequencing (sample Il-1); 2) 3.28 µg total RNA from ∼45 part-fed virgin females for Illumina sequencing (sample Il-2); and 3) 3.25 µg total RNA from ∼30 part-fed virgin females for 454 pyrosequencing (sample 454). RNA yield and purity (260/280) were determined using a Nanodrop 2000 spectrophotometer (Thermofisher, Wilmington, DE). Samples with low purity (<1.8) were discarded.

Illumina sequencing

For Illumina sequencing, samples extracted at ODU were submitted to the North Carolina State University Genome Sciences Laboratory and prepared for sequencing using the Illumina TruSeq RNA Sample Prep Kit v2 (Part No. 15026495, Illumina, Inc. San Diego, CA). The integrity of the individual RNA samples was evaluated using the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA); samples that did not meet minimum requirements (RNA integrity ≥8) were discarded. A minimum of 1.0 µg high quality total RNA was used for Illumina sequencing.

Following PCR amplification, adapters were included for sequencing with paired ends. For paired ends, Illumina GA-II sequencing adapters were ligated to the fragments, as described by Illumina's Paired-End Sample Preparation Guide (catalogue number PE-930-1001).

454 pyrosequencing

For 454 pyrosequencing, the total RNA was thawed and mRNA isolated from each of 3 female synganglion samples, using an Oligotex mRNA isolation kit according to the manufacturer's recommendations. The samples were evaluated using the Bioanalyzer 2100 as described above to insure that they met the minimum requirements for 454 sequencing. Purified mRNA was ethanol precipitated, rehydrated in 2 µl of RNase-free water and combined with 10 pmol of modified 3′ reverse transcription primer (5′-ATTCTAGAGACCGAGGCGGCCGACATGT₍₄₎GT₍₉₎CT₍₁₀₎VN-3′) [35] and 10 pmol SMART IV oligo (5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3′) [36]. The resulting 4 µl were incubated at 72°C for 2 min and then combined with the following reagents on ice: 1 µl RNase Out (40 U/µl, 2 µl 5X first strand buffer, 1 µl 20 mM DTT, 1 µl dNTP mix (10 mM each) and 1 µl Superscript II reverse transcriptase) (Invitrogen, Carlsbad, CA). The reaction was incubated at 42°C for 90 min then diluted to 30 µl with TE buffer (10 mM Tris HCL pH 7.5, 1 mM EDTA) and stored at −20°C until further use. To synthesize second strand cDNA, 5 µl of first-strand cDNA was mixed with 10 pmol of modified 3′ PCR primer (5′-ATTCTAGAGGCCGAGGCGGCCGACATGT₍₄₎GTCT₍₄₎GTTCTGT₍₃₎CT₍₄₎VN-3′) [35], 10 pmol of 5′ PCR primer (5′-AAGCAGTGGTATCAACGCAGAGT-3′) [36], 5 µl 10X reaction buffer, 1 µl dNTP mix, 2 µl MgSO₄, 0.4 µl Platinum HiFi Taq Polymerase and 34.6 µl H₂O (Invitrogen). Thermal cycling conditions were 94°C for 2 min followed by 20 cycles of 94°C for 20 sec, 65°C for 20 sec and 68°C for 6 min. For optimization of the PCR reaction, 5 µl aliquots from cycles 18, 22 and 25 were analyzed on a 1% agarose gel. An additional 5 reactions were carried out with 20 cycles (the optimized number of cycles) to produce sufficient quantities of cDNA for preparation of the 454 library. Following PCR, the contents from the different samples were combined into a single sample and the cDNA was purified using a PCR purification kit (Qiagen) according to the manufacturer's recommendations.

For 454 pyrosequencing, the cDNA library was prepared with the Standard Flex Platform kit (GSLR70 sequencing kit, Cat. No. 04 932 315 001; Roche, Branford CT and Qiagen, Indianapolis, IN) for pyrosequencing on the GS-FLX sequencer according to the manufacturer's recommendations which have been described previously [27], [34], [37]. The only deviation from the protocol was that DNA-positive beads were enriched after emulsification PCR in order to increase the number of reads collected during titration. Enrichment was done so that only beads containing DNA were loaded and the data generated during titration sequencing could also be used in the assembly of contiguous sequences. Enrichment of DNA-positive beads was completed exactly as described by Margulies et al. [37].

Bioinformatics

Assembly was done using CLC-BIO program. We first trimmed the sequences of ambiguous bases, low quality base calls, and by removing any latent primer or adapter sequences. The de novo assembly resulted in ∼41,000 transcripts for sample Il-1 and ∼30,800 transcripts for Il-2 versus 20,600 transcripts for sample 454. Transcripts were identified (annotated) by BLAST [37] against the GenBank nr and EST databases at an E-value of E-06 (or lower) for the Illumina assemblies and E-10 for 454 (although with few exceptions, only matches E-06 or lower were accepted for inclusion in the data tables). Gene Ontology (GO) categorizations of the functional annotations of the top BLASTx hits (1E-06 cutoff) for biological processes (BP) levels 2 and 3 were done using the program Blast2GO [38], [39] in December 2010 for the Illumina transcripts and May, 2011 for the 454 transcripts. Functional assignments were based on an e-6 cut-off (with selected exceptions as justified by other evidence) and conserved domain matches (SMART, KOG and Pfam databases). Additional BLAST searches were done for selected transcripts of interest against the conspecific I. scapularis genome, ver. IscaW1.05.1 (www.vectorbase.org).

Additional evidence to support the annotation of transcripts of interest in the different transcripts was done using alignments with conspecific genes or other arthropod genes. Alignments comparing transcriptome transcripts with the conspecific genome and other species were done using Geneious (Biomatters <system@biomatters.com>) and the alignment programs provided.

With few exceptions, transcripts that showed less than 80% pairwise similarity with the conspecific I. scapularis genome or other species were regarded as unsupported and were removed from the lists of annotated genes.

Data deposition

The Illumina transcriptomes were deposited in the NCBI Sequence Read Archive under project number PRJNA230499. The mixed unfed-fed-replete female synganglion biosample was deposited under sample number Biosample SAMN0249371, accessible with the following link: http://www.ncbi.nlm.nih.gov/biosample/SAMN02429371. The Transcriptome Shotgun Assembly (TSA) project for the part-fed virgin female synganglion assembly was deposited at DDBJ/EMBL/GenBank under accession number GBBN00000000. The version described in this paper is the first version, GBBN01000000. It is accessible with the following link http://www.ncbi.nlm.nih.gov/biosample/SAMN02678957. The Roche 454 pyrosequencing raw reads file for the part-fed virgin female synganglion assembly was deposited in the NCBI Sequence Read Archive under project number PRJNA242857 with SRR # SRR1214480. The 454 transcriptome was deposited in the TSA and will be published pending curation. The transcriptome fasta file is also available in “Supporting Information”.

Results and Discussion

Raw reads, base pairs and assembly

The results of Illumina and 454 sequencing for the three different samples are shown in Table 1. Following sequencing, the raw reads were assembled with the CLC-BIO assembly program (CLC Bio, Cambridge, MA).

Table 1. Statistical summary of results of deep sequencing of female synganglion RNA extracts of the tick, Ixodes scapularis by Illumina and 454 pyrosequencing.

Sample Il-1. Mixed unfed, partfed & replete females by Illumina
Parameter	Count	Average length (bp¹)	Total read length (total bases)
Reads	34,520,330	68	2,346,107,140
Matched	34,273,479	68	2,331,047,269
Not matched	246,851	61	15,059,871
No. Contigs	41,249	480	19,809,620

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bp = base pairs.

NR = Not reported.

For sample Il-1 (mixed females), sequencing yielded a total of 34,520,330 raw reads for a total read length of 2,346,107,140. Almost all reads were matched (34,273,479). The average read length (matched reads) was 68 bp, and included a total length of 2,331,047,269 bp. Following vector trimming, the raw reads were assembled to a total of 41,249 transcripts with an average length of 480 bp, comprising 19,809,620 bp.

For sample Il-2 (part-fed females), sequencing yielded a total of 117,900,476 raw reads. The average length was 72 bp and included a total of 8,488,934,272 bp. However, less than half were matched reads (43,351,571), comprising only 3,121,313,112 bp. Following vector trimming, the raw reads were assembled to a total of 30,838 transcripts with an average length of 655 bp, comprising 20,206,192 bp.

For sample 454 (part-fed females), sequencing yielded a total of 394,946 raw reads. The average length was 268 bp and included a total length of 105,795,232 bp. Following vector trimming, the raw reads were assembled to a total 20,630 transcripts, with an average length of 523 bp, comprising 10,979,742 bp.

BLAST matching of the transcript files showed that almost all of the top hits matched sequences from the I. scapularis genome (Fig. 1). Approximately 17,000 sequences matched similar sequences from I. scapularis, or approximately 91.5% of all matched transcripts. Similar findings were made with BLAST matches from samples Il-1 and 454 (data not shown).

Gene ontology

The program BLAST2GO was used to map the top BLASTx matches (e-value 1e-06) and to assign gene ontology (GO) term annotations [38], [39] in December 2011. For Biological Processes (BioPro) at level 2, GO term searching was highest for sample 454 (46.4%) as compared to only 19.6% and 27.4% for the two Illumina samples. GO term searching success of less than 50% of the expressed genes has been reported in transcriptome studies of other ticks, e.g., the D. variabilis synganglion transcriptome [26] and the D. variabilis male reproductive organs. The GO term assignments for all BioPro level 2 are shown in Fig. 2. Overall, there was little difference in the GO assignments in these transcriptomes, although there were 38 transcripts assigned to reproduction in Il-1(mixed females) as compared to only 8 transcripts in sample Il-2 (part-fed females) versus 85 transcripts in sample 454. Categories considered of greatest interest for regulating synganglion biological activity, development and reproduction in the 3 transcriptomes are shown in Table 2 (BioPro Level 2).

Fig. 2A shows the 19 GO term assignments for sample Il-1 (8,086 transcripts); Fig. 2B shows the 20 GO term assignments for sample IL-2 (8,445 transcripts); Fig. 2C shows the 21 GO term assignments for sample 454 (9,574 transcripts).

Table 2. Gene Ontology categories in three different transcriptomes from samples of the Ixodes scapularis female synganglion: GO terms searching success, similarities and differences.

BioProcess Level 2 (GO term)		% Sample Il-1	% Sample Il-2	% Sample 454
	Metabolic and cellular processes	60.3	69.7	50.4
	Biological regulation	8.9	11.6	9.2
	Signaling	4.0	4.1	4.5
	Response to stimulus	5.7	2.0	6.7
	Developmental processes	1.9	1.3	3.7^*
	Immunity	0.4	0.3	0.8
	Locomotion	0.5	0.2	0.8
	Growth	0.4	0.1	0.7
	Reproduction	0.5	0.1	0.9
	Totals (% all categories)	82.5	89.5	73.2
	Search success: total transcripts/(% all transcripts)	8,086 (19.6%)	8,445 (27.5%)	9,574 (46.4%)
BioProcess Level 2 (GO term)	Metabolic and cellular processes	50.8	46.3	46.5
	Biological regulation	5.3	5.9	7.3
	Oxidation-reduction	4.1	3.3	3.1
	Signaling	0.8	5.2^*	1.3
	Cellular response to stimulus	3.0	0.8^*	3.3
	Response to external stimuli	1.4	1.0	3.0
	Cellular developmental processes	0.5	0.7	1.3^*
	Immunity	0.2	0.3	0.3
	Hormonal metabolic processes	0.6	2.4^*	0.1
	Growth	0.0	0.1	0.2
	Reproduction	0.5	0.3	0.9
	Totals (% all categories)	67.2	66.3	67.3
	Search success: total transcripts/(% all transcripts)	11,740 (28.5%)	23,573 (76.4%)	14,249 (69.1%)
Molecular Level 3 (GO term)	Hydrolase activity	13.1	12.7	11.8
	Transferase activity	12.6	11.9	9.5
	Ion binding	11.7	10.8	8.3
	Nucleotide binding	8.8	9.2	10.7
	Nucleoside binding	0.1	6.8^*	6.5
	Nucleic acid binding	9.4	10.3	7.8
	Tetrapyrrole binding	1.2	0.8	1.0
	Protein binding	8.4^*	10.0^*	9.1
	Carbohydrate binding	0.5	0.5	0.6
	Carboxylic acid binding	0.3	0.2	0.2
	Lipid binding	0.5	0.4	2.0
	Oxireductases activity	6.5	4.8	8.0
	Signal transducer activity	3.4	4.4	2.9
	Neurotransmitter binding	---	0.1	0.1
	Kinase regulatory activity	---	0.1	0.2
	Transmembrane transporter activity	3.0	2.9	2.8
	Nucleoside-triphosphatase activity	1.0	1.0	0.2
	Enzyme inhibitor activity	0.7	0.5	0.8
	Enzyme activator activity	0.6	0.5	0.2
	Totals (selected gene categories)	81.8	65.5	83.3
	Search success: total transcipts/(% all transcripts)	8,160 (19.8%)	17,660 (57.3%)	2,629 (12.74%)

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* Major difference.

For BioPro level 3, there was little difference between samples Il-2 and 454 (76.4% and 69.1%), but both samples had much higher GO term searching success than sample Il-1. GO term searching success for these samples was comparable to studies of transcriptomes of other tick tissues sequenced by 454 pyrosequencing, e.g., the transcriptome of the D. variabilis male reproductive organs (30.2%, [34] and synganglion (29.3%, [26] and the salivary transcriptome of the black-legged tick I. scapularis (66%) [16]. It is likely that the large number of transcripts that could be not be mapped was due to novel and/or unknown sequences which have not been annotated, a phenomenon reported previously for transcriptomes of other tick tissues [16].

The GO term assignments for biological processes at level 3 are shown in Figures 3, 4, 5 and selected categories of interest in Table 2 (BioProcesses Level 3). Many more categories regulating synganglion biological activity were found than were identified in BioPro level 2, including biological regulation, oxidation-reduction, cellular response to stimulus, cell to cell and other signaling types, cellular developmental processes, hormone metabolic processes, immune response and reproduction. Of special interest for sensory perception was the number of messages for responses to external, abiotic and chemical stimuli, representing as much as 1.4% of genes in sample Il-1, 1.0% in sample Il-2 and 3.0% in sample 454. Transcripts matching genes involved in cell to cell signaling, signaling processes, cell to cell communication and signaling pathways represented only 0.8% of total genes in sample Il-1 versus much larger percentages in the other transcriptomes, specifically as much as 5.2% of the total genes in sample Il-2 and 1.3% in sample 454 (Table 2).

This figure shows the 56 GO term assignments categorized in this transcriptome (11,740 transcripts).

This figure shows the 84 GO term assignments categorized in this transcriptome (23,573 transcripts).

This figure shows 81 GO term assignments categorized in this transcriptome (14,249 transcripts).

Comparison of the I. scapularis synganglion 454 transcriptome with the D. variabilis transcriptomes (also created by 454) [26] showed that, for Bioprocesses Level 2, the synganglion of I. scapularis expressed a much higher number of transcripts involved in response to stimulus, biological regulation and developmental processes than similar processes in the synganglion of D. variabilis. Whether this reflects true differences in expression or is a function of the larger proportion of genes mapped in I. scapularis (46.4%) versus the proportion searched in D. variabilis (29.3%) is unknown.

In addition to biological functions, GO term searching also examined the numerous GO categories by molecular function. At molecular level 3, GO term searching of the transcriptome for sample Il-1 showed 34 categories with a total of 8,160 genes, or 19.8% of all transcripts (Fig. 6). The most abundant categories were hydrolase activity (1,069), transferase activity (1,024), ion binding (952), nucleic acid binding (765), nucleotide binding (721), protein binding (681), oxireductase activity (528), and signal transducer activity (275). GO term searching of the transcriptome for sample Il-2 showed 44 categories with a total of 17,660 genes, or 57.3% of all transcripts (Fig. 7). The most abundant categories were hydrolase activity (2,246), transferase activity (2,098), ion binding (1,900), nucleic acid binding (1,815), protein binding (1,762), nucleotide binding (1,625), oxireductase activity (885), and signal transducer activity (771). Of special interest for synganglion regulatory activity was neurotransmitter binding (23 genes) and kinase regulatory activity (18 genes) (highlighted in yellow). In contrast, although the transcriptome for sample 454 showed 58 categories, the total number of genes mapped was only 2,629 genes, or 12.7% of all transcripts (Fig. 8). The most abundant categories were hydrolase activity (309), nucleotide binding (280), transferase activity (250), protein binding (240), ion binding (217), oxireductase activity (210), nucleic acid binding (204), nucleoside binding (171) and signal transducer activity (76). Little evidence of neurotransmitter receptors or neuropeptides was found at this level, namely neurotransmitter receptor binding with 3 genes and peptide binding with 10 genes. However, others may have been present but incorporated into broader categories.

This figure shows the 34 GO assignments categorized in this transcriptome (8,160 transcripts).

This figure shows the 44 GO assignments categorized in this transcriptome (17,660 transcripts).

This figure shows the 58 GO assignments categorized for this transcriptome (2,629 transcripts).

Table 2 shows the contrasts for 19 GO molecular categories of interest for the three different transcriptomes as percentages of the total searched genes. Little difference was noted in the percentages of gene messages in these GO categories among the three different transcriptomes.

Detailed analysis of selected genes/gene categories of interest

Tables 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 compare the three different transcriptomes with respect to the numbers of transcribed genes (i.e., messages), transcript frequency and species matched for 12 gene categories of special interest for understanding synganglion function (<e-06). Transcript frequency indicates the number of transcripts that predicted the same gene identified in GenBank and should not be misconstrued as a measure of gene expression. Variations in transcript frequency may be due to errors in assembly and/or annotations of the same gene in different species. These categories include neuropeptides, neuropeptide receptors, neurotransmitter receptors, other GPCRs, hormone receptors, iron transport/iron storage compounds, immune peptides, reproduction-related compounds, steroid receptor, oxidative or environmental stress compounds and chitin synthesis/cuticle associated compounds.

Table 3. Synopsis of 12 major gene categories in the Ixodes scapularis female synganglion with comparison of the different transcriptomes-neuropeptides.

	Sample Il-1		Sample Il-2		Sample 454
Neuropeptide Name (GO:0005184)^a	No. Trans^b	Species^c	No. Trans^b	Species^c	No. Trans^b	Species^c
Allatostatin	1	I. scapularis	0	-----	1	D. variabilis
Allatotropin	0	-----	1	I. scapularis	1	I. scapularis
Bursicon-α	1	I. scapularis	0	----	0	----
CRF^d	2	I. scapularis	0	----	0	----
CRF^d-binding protein	0	----	2	I. scapularis/C. floridanus	0	----
Eclosion hormone	0	----	1	I. scapularis	0	----
FMRFamide	0	----	1	“	0	----
Glycoprotein A	1	I. scapularis	1	“	0	----
Insulin-like peptide	3	“	1	“	0	----
Ion transport peptide	5	“	2	“	0	----
Myoinhibitory peptide	1	“	0	-----	0	----
Neurophysin-isotocin	1	C. comersonii	1	C. commersonii	0	----
Orcokinin 5	1	I. scapularis	0	-----	0	-----
Sulfakinin	1	D. variabilis	1	I. scapularis	0	-----
Precursor SIFamide	1	I. scapularis	0	I. scapularis	0	-----
No. Neuropeptides/(No Transcripts)	11 (18)		9 (11)		2 (2)

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GO terms are Bioprocesses definitions from the Gene Ontology Consortium (www.geneontology.org).

No. Trans = number of Transcripts. Transcript frequency indicates the number of transcripts that were annotated with the same gene identified in GenBank and should not be misconstrued as a measure of gene expression. If multiple transcripts matched the same GenBank accession number, only the transcript with the highest e-value matching the specific accession number was included. High contig frequency may have been due to errors in assembly and/or annotations of the same gene in different species. Transcripts were annotated as a particular gene message based on strong (i.e., low) e-value and very high % sequence alignment. See Table 13 for representative examples and see text for more detailed descriptions of these methods.

Full names of abbreviated species at the end of Table 12.

Abbreviations of scientific terms: CRF = corticotropin releasing factor.

Total all neuropeptides = 15; Note: in some cases, more than one neuropeptide may result via post-translational modifications from a single prepropeptide mRNA. Transcripts for Pre-protein convertase, an enzyme essential for conversion of neuropeptides to the mature form, also were found.

Table 4. Synopsis of 12 major gene categories in the Ixodes scapularis female synganglion with comparison of the different Transcriptomes – neuropeptide receptors.

	Sample Il-1		Sample Il-2		Sample 454
Receptor Name (GO:0008188)^a	No. Trans^b	Species^c	No. Trans^b	Species^c	No. Trans^b	Species^c
Allatostatin	3	I. scapularis	5	I. scapularis	0	-----
Calcitonin	5	I. scapularis	0	-----	2	I. scapularis
CCAP^d	3	I. scapularis/T. castaneum	1	T. castaneum	0	-----
Corazonin	1	I. scapularis	1	I. scapularis	0	-----
CRF^d	0	-----	4	"	0	-----
Eclosion hormone	1	I. scapularis	1	I. scapularis	0	-----
GnRN/AKH-like^d	1	I. scapularis	1	"	0	-----
Insulin receptor	4	I. scapularis	3	"	0	-----
Neuropeptide F	0	-----	2	"	0	-----
Proctolin	0	-----	0	-----	1	D. pulex
Pyrokinin	0	-----	3	I. scapularis/D. variabilis	0	-----
SIFamide	1	I. scapularis	1	I. scapularis	0	-----
Sulfakinin	0	-----	1	C. quinquefasciatus	0	-----
Tachykinin	0	-----	1	N. vitripennis	1	N. vitripennis
No. Receptors/(No. transcripts)	8 (19)		12 (24)		3 (4)

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Footnotes a and b as in Table 3.

Full names of abbreviated species at the end of Table 12.

Abbreviations of scientific terms: CCAP = Cardioacceleratory peptide; CRF = Corticotropin releasing factor;

GnRH/AKH-like = Gonadotropin releasing hormone/adipokinetic hormone-like.

Table 5. Synopsis of 12 major gene categories in the Ixodes scapularis female synganglion with comparison of the different Transcriptomes –Neurotransmitter receptors and transporters.

	Sample Il-1			Sample Il-2	Sample 454
Receptor Name (GO:0045213)^a	No. Trans^b	Species^c	No. Trans^b	Species^c	No. Trans^b	Species^c
Acetylcholine	10	I. scapularis/others¹	13	I. scapularis/others¹	3	I. scapularis
GABA^d	2	I. scapularis	3	I. scapularis/R. microplus	0	-----
GABA transporter	8	"	7	I. scapularis	5	I. scapularis
Dopamine	5		6	"	1	Gallus gallus
Glutamate (NMDA^d, Ionotropic, Metabotropic)	21	I. scapularis/others¹	24	"	5	I. scapularis/others¹
Na+- neurotransmitter/Symporter	3	I. scapularis	3	"	0	-----
Octopamine	4	"	6	"	2	A. gambiae/others¹
Serotonin	2	I. scapularis	3	"	0	-------
No. Receptors/(No. Transcripts)	8 (55)		8 (65)		5 (16)

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Footnotes a and b as in Table 3; ^c Full names of abbreviated species at the end of Table 12; ^dAbbreviations of scientific terms:

GABA = γ-aminobutyric acid; NMDA = N-methyl-D-aspartate.

Others include: A. gambiae; A. meliffera; A. pisum; A. suum; B. mori; C. floridanus; C. intestinalis; D. rerio; D. melanogaster; G. gallus; H. americanus; Homo sapiens; N. vitripennis; P. humanus; P. pseudoannulata; R. microplus; S. kowalevski; T. verrucosum; T. castaneum.

Table 6. Synopsis of 12 major gene categories in the Ixodes scapularis female synganglion with comparison of the different Transcriptomes –Other GPCR receptors.

	Sample Il-1		Sample Il-2		Sample 454
Receptor Name (GO:0007218^a)	No. Trans^b	Species^c	No. Trans^b	Species^c	No. Trans^b	Species^c
Pheromone odorant receptor	1	I. scapularis	2	I. scapularis	1	I. scapularis
Gustatory receptor	1	N. vitripennis	0	-----	1	N. vitripennis/others
GPCRs unidentified	28	I. scapularis/P. humanus	49	I. scapularis/An. gambiae	1	S. purpuratus/others¹
No. GPCRs/(No. Transcripts)	3 (30)		2 (51)		3 (3)

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Footnotes a and b as in Table 3; ^c Full names of abbreviated species at the end of Table 12; ¹Others include: N. vitripennis; T. castaneum; D. pulex.