Abstract
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 β 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.
Highlights
Ticks are blood-feeding parasites that serve as vectors of the causative agents of many important diseases affecting humans and animals, e.g., Lyme disease, Rocky Mountain spotted fever, tickborne encephalitis, anaplasmosis, babesiosis and many others [1]
Ten major Gene Ontology (GO) categories were identified in this male reproductive cDNA library as follows: reproduction-related functions, peptidases, proteinase inhibitors, hydrolases, lipases, oxidative stress proteins, environmental stress proteins, innate immune proteins, cellular adhesion-cell surface modulating proteins and signal transduction receptors (GPCRs)
Proteins were identified by LC MS/MS in the spermatophore and male accessory gland (MAG)/testis/vas deferens (TVD); excluding metabolic, ribosomal and mitochondrial proteins, 42 of the 56 proteins identified by this method were found in the male accessory gland/testis vas deferens (MAG/TVD) transcriptome
Summary
Ticks are blood-feeding parasites that serve as vectors of the causative agents of many important diseases affecting humans and animals, e.g., Lyme disease, Rocky Mountain spotted fever, tickborne encephalitis, anaplasmosis, babesiosis and many others [1]. In the hard ticks (Ixodidae), blood-feeding stimulates oogenesis and spermatogenesis. Blood-feeding stimulates females to secrete sex pheromones that attract sexually active males for mating and insemination. Males rapidly assemble their spermatophores which fill with spermatids and seminal fluid [2]. Little is known about the composition of the spermatophores or how they are assembled. In females, mating and copulation induces the transition from slow to rapid feeding, followed by the cascade of reproductive events that eventually results in egg production. The identification of the male pheromone that triggers these profound physiological changes in the female of Amblyomma hebraeum was voraxin, consisting of two engorgement factor proteins [3]; to date, this function has not been confirmed in other tick species
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