RESUMEN
Genomes of ticks display reductions, to various extents, in genetic coding for enzymes of the haem biosynthetic pathway. Here, we mined available transcriptomes of soft tick species and identified transcripts encoding only half of the enzymes involved in haem biosynthesis. Transcripts identified across most species examined were those coding for porphobilinogen synthase, coproporphyrinogen oxidase, protoporphyrinogen oxidase, and ferrochelatase. Genomic retention of porphobilinogen synthase seems to be soft tick-restricted as no such homologue has been identified in any hard tick species. Bioinformatic mining is thus strongly indicative of the lack of biochemical capacity for de novo haem biosynthesis, suggesting a requirement for dietary haem. In the hard tick Ixodes ricinus, depletion of dietary haem, i.e. serum feeding, leads to oviposition of haem-free eggs, with no apparent embryogenesis and larvae formation. In this work, we show that serum-fed Ornithodoros moubata females, unlike those of I. ricinus, laid haem-containing eggs similarly to blood-fed controls, but only by a small proportion of the serum-fed females. To enhance the effect of dietary haem depletion, O. moubata ticks were serum-fed consecutively as last nymphal instars and females. These females laid eggs with profoundly reduced haem deposits, confirming the host origin of the haem. These data confirm the ability of soft ticks to take up and allocate host haem to their eggs in order to drive reproduction of the ticks.
Asunto(s)
Argasidae , Ixodidae , Ornithodoros , Animales , Femenino , Hemo , Porfobilinógeno SintasaRESUMEN
In addition to being vectors of pathogenic bacteria, ticks also harbor intracellular bacteria that associate with ticks over generations, aka symbionts. The biological significance of such bacterial symbiosis has been described in several tick species but its function in Ixodes ricinus is not understood. We have previously shown that I. ricinus ticks are primarily inhabited by a single species of symbiont, Midichloria mitochondrii, an intracellular bacterium that resides and reproduces mainly in the mitochondria of ovaries of fully engorged I. ricinus females. To study the functional integration of M. mitochondrii into the biology of I. ricinus, an M. mitochondrii-depleted model of I. ricinus ticks was sought. Various techniques have been described in the literature to achieve dysbiosed or apo-symbiotic ticks with various degrees of success. To address the lack of a standardized experimental procedure for the production of apo-symbiotic ticks, we present here an approach utilizing the ex vivo membrane blood feeding system. In order to deplete M. mitochondrii from ovaries, we supplemented dietary blood with tetracycline. We noted, however, that the use of tetracycline caused immediate toxicity in ticks, caused by impairment of mitochondrial proteosynthesis. To overcome the tetracycline-mediated off-target effect, we established a protocol that leads to the production of an apo-symbiotic strain of I. ricinus, which can be sustained in subsequent generations. In two generations following tetracycline administration and tetracycline-mediated symbiont reduction, M. mitochondrii was gradually eliminated from the lineage. Larvae hatched from eggs laid by such M. mitochondrii-free females repeatedly performed poorly during blood-feeding, while the nymphs and adults performed similarly to controls. These data indicate that M. mitochondrii represents an integral component of tick ovarian tissue, and when absent, results in the formation of substandard larvae with reduced capacity to blood-feed.
Asunto(s)
Ixodes , Animales , Femenino , Ixodes/microbiología , Tetraciclina , Antibacterianos , Mitocondrias , SimbiosisRESUMEN
During feeding on vertebrate hosts, ticks secrete saliva composed of a rich cocktail of bioactive molecules modulating host immune responses. Although most of the proteinaceous fraction of tick saliva is of little immunogenicity, repeated feeding of ticks on mammalian hosts may lead to impairment of tick feeding, preventing full engorgement. Here, we challenged rabbits with repeated feeding of both Ixodes ricinus nymphs and adults and observed the formation of specific antibodies against several tick salivary proteins. Repeated feeding of both I. ricinus stages led to a gradual decrease in engorged weights. To identify the salivary antigens, isolated immunoglobulins from repeatedly infested rabbits were utilized for a protein pull-down from the saliva of pilocarpine-treated ticks. Eluted antigens were first identified by peptide mass fingerprinting with the aid of available I. ricinus salivary gland transcriptomes originating from early phases of tick feeding. To increase the authenticity of immunogens identified, we also performed, for the first time, de novo assembly of the sialome from I. ricinus females fed for six days, a timepoint used for pilocarpine-salivation. The most dominant I. ricinus salivary immunogens identified in our study were zinc-dependent metalloproteases of three different families. To corroborate the role of metalloproteases at the tick/host interface, we fed ticks micro-injected with a zinc metalloprotease inhibitor, phosphoramidon, on a rabbit. These ticks clearly failed to initiate feeding and to engorge. However, neither feeding to ticks immune blood of repeatedly infested rabbits, nor phosphoramidon injection into ticks, prevented their engorgement when fed in vitro on an artificial membrane system. These data show that Zn metalloproteases play a decisive role in the success of tick feeding, mediated by complex molecular interactions between the host immune, inflammatory, and hemostatic processes, which are absent in in vitro feeding. This basic concept warrants further investigation and reconsideration of the current strategies towards the development of an effective "anti-tick" vaccine.
Asunto(s)
Ixodes , Infestaciones por Garrapatas , Animales , Proteínas de Artrópodos , Femenino , Metaloproteasas , Conejos , Glándulas Salivales , Proteínas y Péptidos SalivalesRESUMEN
Blood-feeding parasites are inadvertently exposed to high doses of potentially cytotoxic haem liberated upon host blood digestion. Detoxification of free haem is a special challenge for ticks, which digest haemoglobin intracellularly. Ticks lack a haem catabolic mechanism, mediated by haem oxygenase, and need to dispose of vast majority of acquired haem via its accumulation in haemosomes. The knowledge of individual molecules involved in the maintenance of haem homeostasis in ticks is still rather limited. RNA-seq analyses of the Ixodes ricinus midguts from blood- and serum-fed females identified an abundant transcript of glutathione S-transferase (gst) to be substantially up-regulated in the presence of red blood cells in the diet. Here, we have determined the full sequence of this encoding gene, ir-gst1, and found that it is homologous to the delta-/epsilon-class of GSTs. Phylogenetic analyses across related chelicerates revealed that only one clear IrGST1 orthologue could be found in each available transcriptome from hard and soft ticks. These orthologues create a well-supported clade clearly separated from other ticks' or mites' delta-/epsilon-class GSTs and most likely evolved as an adaptation to tick blood-feeding life style. We have confirmed that IrGST1 expression is induced by dietary haem(oglobin), and not by iron or other components of host blood. Kinetic properties of recombinant IrGST1 were evaluated by model and natural GST substrates. The enzyme was also shown to bind haemin in vitro as evidenced by inhibition assay, VIS spectrophotometry, gel filtration, and affinity chromatography. In the native state, IrGST1 forms a dimer which further polymerises upon binding of excessive amount of haemin molecules. Due to susceptibility of ticks to haem as a signalling molecule, we speculate that the expression of IrGST1 in tick midgut functions as intracellular buffer of labile haem pool to ameliorate its cytotoxic effects upon haemoglobin intracellular hydrolysis.