Exploring the Neuropeptides, Neuropeptide Receptors and Neurotransmitter Receptors in the Synganglia of Part-Fed Females of \u3ci\u3eOrnithodoros turicata\u3c/i\u3e (Argasidae) and \u3ci\u3eIxodes scapularis\u3c/i\u3e (Ixodidae) with Insights Into Their Roles in Contrasting Biology

The neurobiology of the synganglion (central nervous system) of the Lyme disease tick, Ixodes scapularis and the soft tick Ornithodoros turicata was evaluated using Illumina GAII high throughput sequencing which generated high coverage cDNA libraries (transcriptomes). These ticks exhibit different biological patterns of feeding, blood meal water, and salt elimination, cuticle plasticity versus cuticle synthesis, development and reproduction. RNA sequencing of I. scapularis, and Ornithodoros turicata yielded a total of 117,900,476 raw reads which were assembled to 30,838 contigs and a total of 63,528,102 also assembled to 132,258 contigs, respectively. Comparison of Gene Ontology (GO) mapping success for genes in 32 important GO molecular categories showed little difference between the two species. Functional assignments of transcripts predicting neuropeptides, neuropeptide receptors and neurotransmitter receptors was done, supported by strong e-values (\u3c -6), and high consensus sequence alignments. For the synganglion of I. scapularis, transcripts predicting 23 neuropeptides and/or their receptors were identified. For the synganglion of O. turicata, 25 neuropeptides and/or their receptors were identified. Both species had transcripts predicting all of the same neuropeptides and/or their neuropeptide receptors in common except for allatotropin peptide, found only in I. scapularis, and allatostatin C, bursicon β, and glycoprotein B, which were found only in O. turicata. If the repertoire of neuropeptide and neurotransmitter messages expressed in the synganglia of O. turicata and I scapularis is so similar, how can we explain the very different physiological processes that occur in these two very different tick species? Real time PCR assays were used to study the expression of candidate genes in response to blood feeding. My study shows a strong similarity in gene identity (annotation/alignments) of both species but marked differences in the gene expression, extent of up-regulation or down-regulation, and the timing of their expression in response to feeding. This may indeed help explain many of the differences in the biology of the two different species. The diversity of messages predicting important genes identified in this study and differences in their expression in response to feeding offers a valuable resource useful for understanding how the tick synganglion regulates important physiological functions in ticks

Using an in vitro System for Maintaining Varroa Destructor Mites on Apis Mellifera Pupae as Hosts: Studies of Mite Longevity and Feeding Behavior

Varroa destructor mites (varroa) are ectoparasites of Apis mellifera honey bees, and the damage they inflict on hosts is likely a causative factor of recent poor honey bee colony performance. Research has produced an arsenal of control agents against varroa mites, which have become resistant to many chemical means of their control, and other means have uncertain efficacy. Novel means of control will result from a thorough understanding of varroa physiology and behavior. However, robust knowledge of varroa biology is lacking; mites have very low survivability and reproduction away from their natural environment and host, and few tested protocols of maintaining mites in vitro are available as standardized methods for varroa research. Here, we describe the \u27varroa maintenance system\u27 (VMS), a tool for maintaining in vitro populations of varroa on its natural host, and present best practices for its use in varroa and host research. Additionally, we present results using the VMS from research of varroa and host longevity and varroa feeding behavior. Under these conditions, from two trials, mites lived an average of 12 and 14 days, respectively. For studies of feeding behavior, female mites inflicted wounds located on a wide range of sites on the host\u27s integument, but preferred to feed from the host\u27s abdomen and thorax. Originally in the phoretic-phase, female mites in VMS had limited reproduction, but positive instances give insights into the cues necessary for initiating reproduction. The VMS is a useful tool for laboratory studies requiring long-term survival of mites, or host-parasite interactions

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

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 (\u3c-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

Mevalonate-Farnesal Biosynthesis in Ticks: Comparative Synganglion Transcriptomics and a New Perspective

Juvenile hormone (JH) controls the growth, development, metamorphosis, and reproduction of insects. For many years, the general assumption has been that JH regulates tick and other acarine development and reproduction the same as in insects. Although researchers have not been able to find the common insect JHs in hard and soft tick species and JH applications appear to have no effect on tick development, it is difficult to prove the negative or to determine whether precursors to JH are made in ticks. The tick synganglion contains regions which are homologous to the corpora allata, the biosynthetic source for JH in insects. Next-gen sequencing of the tick synganglion transcriptome was conducted separately in adults of the American dog tick, Dermacentor variabilis, the deer tick, Ixodes scapularis, and the relapsing fever tick, Ornithodoros turicata as a new approach to determine whether ticks can make JH or a JH precursor. All of the enzymes that make up the mevalonate pathway from acetyl-CoA to farnesyl diphosphate (acetoacetyl-CoA thiolase, HMG-S, HMG-R, mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase, and farnesyl diphosphate synthase) were found in at least one of the ticks studied but most were found in all three species. Sequence analysis of the last enzyme in the mevalonate pathway, farnesyl diphosphate synthase, demonstrated conservation of the seven prenyltransferase regions and the aspartate rich motifs within those regions typical of this enzyme. In the JH branch from farnesyl diphosphate to JH III, we found a putative farnesol oxidase used for the conversion of farnesol to farnesal in the synganglion transcriptome of I. scapularis and D. variabilis. Methyltransferases (MTs) that add a methyl group to farnesoic acid to make methyl farnesoate were present in all of the ticks studied with similarities as high as 36% at the amino acid level to insect JH acid methyltransferase (JHAMT). However, when the tick MTs were compared to the known insect JHAMTs from several insect species at the amino acid level, the former lacked the farnesoic acid binding motif typical in insects. The P450s shown in insects to add the C10,11 epoxide to methyl farnesoate, are in the CYP15 family; this family was absent in our tick transcriptomes and in the I. scapularis genome, the only tick genome available. These data suggest that ticks do not synthesize JH III but have the mevalonate pathway and may produce a JH III precursor

First Transcriptome of the Testis-Vas Deferens-Male Accessory Gland and Proteome of the Spermatophore from Dermacentor variabilis (Acari: Ixodidae)

Ticks are important vectors of numerous human diseases and animal diseases. Feeding stimulates spermatogenesis, mating and insemination of male factors that trigger female reproduction. The physiology of male reproduction and its regulation of female development are essentially a black box. Several transcriptomes have catalogued expression of tick genes in the salivary glands, synganglion and midgut but no comprehensive investigation has addressed male reproduction and mating. Consequently, a new global approach using transcriptomics, proteomics, and quantitative gene expression is needed to understand male reproduction and stimulation of female reproduction

Gene Ontology (GO) term assignments at biological processes level 3 for the transcriptome for sample Il-2 assembled following sequencing by Illumina.

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

Synopsis of 12 major gene categories in the <i>Ixodes scapularis</i> female synganglion with comparison of the different Transcriptomes –Hormone/other steroid proteins and receptors.

<p>Synopsis of 12 major gene categories in the <i>Ixodes scapularis</i> female synganglion with comparison of the different Transcriptomes –Hormone/other steroid proteins and receptors.</p

Blast matching of contig files in the transcriptomes showing the top hit species distribution.

<p>Blast matching of contig files in the transcriptomes showing the top hit species distribution.</p

Synopsis of 12 major gene categories in the <i>Ixodes scapularis</i> female synganglion with comparison of the different Transcriptomes – neuropeptide receptors.

<p>Synopsis of 12 major gene categories in the <i>Ixodes scapularis</i> female synganglion with comparison of the different Transcriptomes – neuropeptide receptors.</p

Statistical summary of results of deep sequencing of female synganglion RNA extracts of the tick, <i>Ixodes scapularis</i> by Illumina and 454 pyrosequencing.

<p>Statistical summary of results of deep sequencing of female synganglion RNA extracts of the tick, <i>Ixodes scapularis</i> by Illumina and 454 pyrosequencing.</p