Tick Haller's Organ, a New Paradigm for Arthropod Olfaction: How Ticks Differ from Insects.

Ticks are the vector of many human and animal diseases; and host detection is critical to this process. Ticks have a unique sensory structure located exclusively on the 1st pairs of legs; the fore-tarsal Haller's organ, not found in any other animals, presumed to function like the insect antennae in chemosensation but morphologically very different. The mechanism of tick chemoreception is unknown. Utilizing next-generation sequencing and comparative transcriptomics between the 1st and 4th legs (the latter without the Haller's organ), we characterized 1st leg specific and putative Haller's organ specific transcripts from adult American dog ticks, Dermacentor variabilis. The analysis suggested that the Haller's organ is involved in olfaction, not gustation. No known odorant binding proteins like those found in insects, chemosensory lipocalins or typical insect olfactory mechanisms were identified; with the transcriptomic data only supporting a possible olfactory G-protein coupled receptor (GPCR) signal cascade unique to the Haller's organ. Each component of the olfactory GPCR signal cascade was identified and characterized. The expression of GPCR, Gαo and ß-arrestin transcripts identified exclusively in the 1st leg transcriptome, and putatively Haller's organ specific, were examined in unfed and blood-fed adult female and male D. variabilis. Blood feeding to repletion in adult females down-regulated the expression of all three chemosensory transcripts in females but not in males; consistent with differences in post-feeding tick behavior between sexes and an expected reduced chemosensory function in females as they leave the host. Data are presented for the first time of the potential hormonal regulation of tick chemosensation; behavioral assays confirmed the role of the Haller's organ in N,N-diethyl-meta-toluamide (DEET) repellency but showed no role for the Haller's organ in host attachment. Further research is needed to understand the potential role of the GPCR cascade in olfaction.

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.

Genomic insights into the Ixodes scapularis tick vector of Lyme disease.

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick-host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host 'questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.

Polymers for the stabilization and delivery of proteins topically and per os to the insect hemocoel through conjugation with aliphatic polyethylene glycol.

Co-feeding of aliphatic polyethylene glycol (PEG), phospholipase A2, anionic and ionic detergents, and amphipathic glycoside with bovine serum albumin (BSA) as a model protein to fourth stadium tobacco budworms, Heliothis virescens, did not affect the levels of BSA in the hemolymph. Covalent conjugation of small proteins like the decapeptide trypsin modulating oostatic factor (TMOF) to polyethylene glycol was previously shown to protect the peptide from protease attack and enhance its accumulation in the insect hemocoel. Whether this polymer chemistry could do the same for larger proteins was examined. The chemistry for the synthesis of polydispersed aliphatic PEG350-insulin and monodispersed aliphatic PEG333-insulin are described herein. Insulin was used for this synthesis and not BSA to better control conjugation among the available free amine groups. When PEGylated insulin or free insulin were fed in artificial diet to fifth stadium budworms, greater concentrations of insulin using the PEGylated variants were found in the hemolymph than when free insulin was used (a 6.7 and 7.3-fold increase for the PEG350 and PEG333 conjugates, respectively). When insulin is topically applied to the dorsum of H. virescens, no insulin is found in the hemolymph. However, after topical application of the PEGylated insulins, PEG350-insulin and PEG333-insulin were detected in the hemolymph. After injections of insulin into the hemocoel of fourth stadium H. virescens, insulin is completely cleared from the hemolymph in 120min. In comparison, PEG350-insulin and PEG333-insulin were present in the hemolymph for 300 and 240min after injection, respectively, translating to a 3.3 and 2.7-fold increase in the length of time insulin remains in the hemolymph after injection.

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 (<-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.

Enhanced activity of an insecticidal protein, trypsin modulating oostatic factor (TMOF), through conjugation with aliphatic polyethylene glycol.

BACKGROUND: Trypsin modulating oostatic factor (TMOF), a decapeptide (Tyr-Asp-Pro-Ala-Pro(6)) isolated from the ovaries of the adult yellow fever mosquito, Aedes aegypti, regulates trypsin biosynthesis. TMOF per os is insecticidal to larval mosquitoes and a good model for the development of technologies to enhance protein insecticide activity by reduced catabolism and/or enhanced delivery to the target. RESULTS: TFA-TMOF-K (TFA = trifluoro acetyl) allowed the specific conjugation of monodispersed, aliphatic polyethylene glycol (PEG) to the amino group of lysine-producing TMOF-K-methyl(ethyleneglycol)(7)-O-propionyl (TMOF-K-PEG(7) P). The addition of lysine to TMOF reduced its per os larval mosquitocidal activity relative to the parent TMOF, but conjugation of TMOF-K with methyl(ethyleneglycol)(7)-O-propionyl increased its toxicity 5.8- and 10.1-fold above that of TMOF and TMOF-K for Ae. aegypti. Enhanced insecticidal activity was also found for larval Ae. albopictus and for neonates of Heliothis virescens and Heliocoverpa zea. Only TMOF-K was found by MS/MS in the hemolymph for H. virescens fed on TMOF-K-PEG(7) P. No TMOF, TMOF-K or PEGylated TMOF-K was detected in the hemolymph after topical applications. CONCLUSIONS: This research suggests that aliphatic PEG polymers can be used as a new method for increasing the activity of insecticidal proteins.

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.This first transcriptome to the reproductive biology of fed male ticks, Dermacentor variabilis, was obtained by 454 pyrosequencing (563,093 reads, 12,804 contigs). Gene Ontology (Biological Processes level III) recognized 3,866 transcripts in 73 different categories; spermiogenesis; spermatogenesis; peptidases, lipases and hydrolases; oxidative and environmental stress; immune defense; and protein binding. Reproduction-associated genes included serine/threonine kinase, metalloendoproteinases, ferritins, serine proteases, trypsin, cysteine proteases, serpins, a cystatin, GPCR and others. qRT-PCR showed significant upregulation from unfed versus fed adult male reproductive organs of zinc metalloprotease, astacin metalloprotease and serine protease, enzymes important in spermiogenesis and mating activity in insects, as well as a GPCR with the greatest similarity to a SIFamide receptor known to be important in regulating courtship behavior in Drosophila. Proteomics on these organs and the spermatophore by tryptic digestion/Liquid chromatography/Mass spectrometry/Mass spectrometry (LC/MS/MS) demonstrated expression of many of the same messages found by 454 sequencing, supporting their identification, and revealed differences in protein distribution in the reproductive system versus the spermatophore. We found Efα but no EF ß in the transcriptome and neither of these proteins in the spermatophore. Thus, the previously described model for male regulation of female reproduction may not apply to other ticks. A new paradigm is needed to explain male stimulation of female tick reproduction.

Comparative efficacy of BioUD to other commercially available arthropod repellents against the ticks Amblyomma americanum and Dermacentor variabilis on cotton cloth.

BioUD is an arthropod repellent that contains the active ingredient 2-undecanone originally derived from wild tomato plants. Repellency of BioUD was compared with five commercially available arthropod repellents against the ticks Amblyomma americanum (L.) and Dermacentor variabilis Say in two-choice bioassays on treated versus untreated cotton cheesecloth. Overall mean percentage repellency against both species was greatest for and did not differ significantly between BioUD (7.75% 2-undecanone) and products containing 98.1% DEET, 19.6% IR3535, and 30% oil of lemon eucalyptus. Products containing 5% and 15% Picaridin and 0.5% permethrin were also repellent compared with untreated controls but to a lesser degree than BioUD. The four most active repellents at the same concentrations used before were directly compared in head-to-head bioassays on cotton cheesecloth. BioUD provided significantly greater overall mean percentage repellency than IR3535 for A. americanum and D. variabilis. BioUD was significantly more repellent than oil of lemon eucalyptus for A. americanum but did not differ significantly in repellency against D. variabilis. No statistically significant difference in overall mean percentage repellency was found between BioUD and DEET for A. americanum or D. variabilis. In a 7-week time course bioassay, BioUD applied to cotton cheesecloth and held at room temperature provided 5 weeks of > 90% repellency against A. americanum.