BACKGROUND
ticks are hematophagous arthropods, notorious as vectors of human and animal pathogens  <cit> .
diseases transmitted by ticks are global medical and veterinary public health problems  <cit> .
ixodes scapularis, known commonly as the deer tick or blacklegged tick, is the major vector for lyme disease, babesiosis, and granulocytic anaplasmosis to humans and domestic animals in the united states.
ticks and other blood-feeding arthropod vectors manipulate host hemostatic and immune responses by secreting molecules from their multifunctional salivary glands.
blood feeding by ticks requires prolonged contact with host tissues and blood, and it has been suggested that the adaptation of ticks to their natural host has resulted in evolution of an appropriate set of salivary components allowing the tick to evade host immunity and prevent coagulation at the feeding site to successfully obtain its blood meal  <cit> .
the blood feeding cycle of larval and nymphal ixodes scapularis typically extends for three to four days, while that of the adult female blacklegged tick lasts approximately six days; during this time the tick alternately secretes salivary fluid into the host and takes up blood from the host  <cit> .
successful blood feeding must require endogenous signalling molecules within the tick to turn on and off salivation and control the release of some  <dig> secreted salivary proteins into the saliva  <cit> .
one possible strategy for reducing tick-transmitted disease incidence could involve manipulating tick salivary secretion so as to interrupt or shorten the duration of host attachment or blood feeding.
accordingly, this research is focussed on elucidating biochemical pathways of two sulfotransferase genes that may be involved in tick salivation and pathogen transmission.
sulfotransferases catalyze transfer of a sulfonyl moiety  from the universal donor 3'-phosphoadenosine-5'-phosphosulfate  to an oxygen or nitrogen acceptor, resulting in production of a sulfate ester.
historically, sulfotransferases have been classified by their sub-cellular localization as either cytosolic or membrane bound  <cit> .
in general, cytosolic sulfotransferases accept relatively small molecules as substrates, while the membrane bound sulfotransferases utilize macromolecules  <cit> .
although a number of sulfotransferase genes are registered in various invertebrate genome databases, only a few invertebrate sulfotransferases have been characterized.
cytosolic sulfotransferases have been characterized from the nematode caenorhabditis elegans  <cit>  and from the arthropods bombyx mori  <cit> , spodoptera frugiperda  <cit> , and drosophila melanogaster  <cit> .
c. elegans contains a glycosaminoglycan membrane bound sulfotransferase with heparan-2-o-sulfotransferase activity  <cit> .
membrane bound tyrosylprotein sulfotransferases have been characterized from drosophila melanogaster and from c. elegans  <cit> .
specifically in salivary glands, tyrosylprotein sulfotransferases have been characterized from human saliva  <cit>  and rat salivary gland tissue  <cit> .
thus, precedent exists for both cytosolic and membrane-localized sulfotransferases in arthropods, and in mammalian salivary glands.
sulfotransferase enzymes modulate the activity of many hormones and proteins; in humans and other mammalian species, some are involved in regulating immune responses and blood coagulation  <cit> .
recently, we predicted that dopamine and octopamine could serve as substrates for tick-derived sulfotransferases  <cit> .
dopamine is known to stimulate secretion of tick saliva through a neurochemical mechanism.
tick salivary fluid secretion is controlled via a dopamine d <dig> receptor and camp dependent protein phosphorylation cascade following salivary gland stimulation by dopamine released from nerve endings  <cit> .
moreover, endogenous dopamine has been identified in salivary glands of the ticks rhipicephalus microplus and amblyomma hebraeum  <cit> .
octopamine also exhibits neuromodulatory functions in arthropods  <cit> .
thus, precedent exists for sulfotransferase enzymes to impact host hemostasis as well as modulate tick salivary or host components which regulate salivation, immune response, or blood coagulation.
our recent report showed the three dimensional structural conservation of two novel tick sulfotransferases  and its ligand docking.
predictions from the modelling were tested and confirmed using native tissue enzyme homogenates from larval and nymphal stage blacklegged ticks  <cit> .
in this present study, we identify the genetic expression of two cytosolic sulfotransferases throughout the tick life cycle and characterize the expressed recombinant proteins.
interestingly, we observed differential gene expression between the two sulfotransferases during blood feeding, and that their recombinant enzymes effectively sulfate neurotransmitters dopamine and octopamine.
taken together, the observed differential expression of these two sulfotransferase genes and the resulting protein affinity for neurotransmitters involved in tick salivation suggests that ixosc sult  <dig> and sult  <dig> may play roles in tick salivation and pathogen transmission processes.
RESULTS
amino acid sequence comparison
the sequence alignment of the two ixodes sulfotransferases  and related mammalian cytosolic sulfotransferases are shown in figure  <dig>  the sult_motif on figure  <dig> indicates the residues which make up the cytosolic sulfotransferase amino acid sequence motif regions.
thus, the amino acid sequence comparison confirms that there is presence of conserved domains of cytosolic sulfotransferases on both ixosc sult  <dig> and  <dig>
transcriptional expression of ixosc sult  <dig> and sult  <dig> genes and gene products
to shed light on the transcriptional control of the two genes during different life stages of i. scapularis, we used real-time quantitative rt-pcr to amplify rna isolated from pooled unfed and blood-fed whole larvae and nymphs, as well as the salivary glands and midguts dissected from both unfed and blood-fed adult stage ticks.
usually, the feeding time varied between  <dig> to  <dig> hours.
expression levels were normalized using the constitutively expressed β-actin transcript as a standard.
results showed that the sult  <dig> was increased during feeding  <dig>  fold in larvae and  <dig>  fold in nymphs.
in contrast, sult  <dig> was decreased during feeding by  <dig>  fold in larvae and  <dig>  fold in nymphs as shown in figure 2a and 2b.
furthermore, the difference in transcript abundance for sult  <dig> and sult  <dig> were statistically significant, p <  <dig>  for sult  <dig> and p <  <dig>  for sult  <dig> during larval stages between unfed and fed, where as p <  <dig>  and p <  <dig>  for sult  <dig> during nymphal stages between unfed and fed, respectively .
briefly, as feeding initiated in larval ticks, sult  <dig> expression increased whereas sult  <dig> expression decreased during feeding in both larval and nymphal stages.
during the adult tick feeding, there was no significant difference in sult  <dig> or sult  <dig> transcript abundance .
similar results were observed at the protein level using western blot probed with antibodies raised against expressed and affinity-purified ixosc sult  <dig> or sult  <dig>  band intensities were quantified using kodak digital science 1d image analysis software and plotted against different tick stages .
values are expressed as mean ± sd for three replicated experiments and show significant differences between blood-fed and unfed states, respectively, at both the nymphal and larval stages.
similar to the quantitative pcr results, the ixosc sult  <dig> and  <dig> protein levels in the adult stage showed no difference on feeding .
full length ixosc sult  <dig> and sult  <dig> were cloned into the ptrchis <dig> topo ta expression vector.
the expressed proteins were purified using a his-select ilap  column.
the purified ixosc sult  <dig> and sult  <dig> showed molecular weights of  <dig> and  <dig> kda, respectively , and the purified proteins were sequenced using liquid automatic sequencing.
the resulting sequences from the expert protein analysis system  confirmed that the expressed proteins are from tick sulfotransferase genes .
enzymatic characterization of ixosc sult  <dig>  and sult  <dig>
we tested whether expressed purified ixosc sult  <dig>  or sult  <dig>  could sulfonate selected potentially relevant substrates.
the rationale for including p-nitrophenol, 17b-estradiol, pregnenolone, and dopamine was that they are prototype substrates for the mammalian sulfotransferases.
the rationale for including octopamine and an additional rationale for including dopamine was based on the relevance of these compounds as arthropod neuromodulators and on the previous structure modelling study  <cit>  which predicted these monoamines as potential substrates for the i. scapularis sulfotransferases.
arthropod steroids such as ecdysone were not tested as substrates, as the previous structure modelling study indicated that these steroid structures are not complimentary to the binding pocket of the i. scapularis sulfotransferases and should not be substrates  <cit> .
each potential substrate was added to  <dig> μm pap35s  and purified enzyme , and was incubated at 37°c.
because of the zwitterionic nature of two of the expected products  at acidic ph, we used cellulose thin-layer chromatography under strongly basic conditions for the separation.
as shown in figure  <dig> and quantified in table  <dig>  p-nitrophenol, dopamine, and r,s-octopamine were substrates for both ixosc sult  <dig>  and sult  <dig> .
the enzyme activity of expressed ixosc sult  <dig>  was found to be  <dig> - <dig>  nmol min- <dig> mg- <dig> and ixosc sult  <dig>  to be  <dig> - <dig>  nmol min- <dig> mg- <dig>  respectively, when incubated with  <dig> μm of each potential substrate.
product formation in the absence of enzyme  was found to be zero.
17β-estradiol and pregnenolone showed no product formation under the conditions tested .
because the 35s-labeled products of dopamine, octopamine, and p-nitrophenol migrated with a similar retention factor , their individual formation was confirmed by mass spectral analysis.
the sulfated product of dopamine and octopamine, respectively, were positively identified by mass spectral detection under negative ionization conditions  .
p-nitrophenylsulfate ion was detected in parallel incubations with both ixosc sult  <dig> and  <dig>   and sult  <dig> .
tlc separation of 35s-labeled product from pap35s  after incubation of  <dig> μm of noted compounds with expressed purified ixosc sult  <dig>  or sult  <dig> as described in experimental procedures.
a substrates  were incubated with pap35s  and purified ixosc sult  <dig>  or ixosc sult  <dig>  for  <dig> min.
product formation in the absence of enzyme  was found to be zero.
discussion
in this study, we describe the expression of novel ixodes scapularis sulfotransferase genes throughout the blacklegged tick life cycle and different states of blood feeding, and characterize their expressed recombinant proteins.
ixosc sult  <dig> and sult  <dig> are expressed during every life stage of blacklegged ticks, and interestingly, mrna and protein levels are moderately but significantly changed  upon blood-feeding at the nymphal and larval stages.
we find it especially interesting that the process of feeding has an opposite effect on relative expression of the ixosc sult  <dig> and sult  <dig> during both the larval and nymphal life stages, although no change in expression levels could be detected in the adult stage salivary gland or midgut .
thus, these two proteins seem to have different roles in the tick salivation and feeding process.
ixosc sult  <dig> mrna and protein were increased upon feeding from a low basal level at the larval and nymphal stage.
in contrast, ixosc sult  <dig> mrna and protein were decreased upon feeding from a high basal level at the larval and nymphal stage.
quantitative rt-pcr and quantification of band intensities by kodak digital analyser confirmed the statistical increase of sult  <dig> and decrease of sult  <dig> as well as their gene products during feeding.
our recent publication  <cit>  on homology modelling, molecular docking and enzyme kinetic studies of sulfotransferase activity from native tick tissues showed similar findings using specific tick homogenates.
it should be noted that these results were obtained from three pools of tissue derived from multiple ticks, and that the blood-fed ticks were sampled after being attached to hosts for  <dig> -  <dig> hours.
because tick salivation and secretion is a dynamic process throughout the multi-day feeding, experiments are in progress to profile the expression during more exact time periods.
while there is evidence for altered expression of multiple genes during tick feeding  <cit> , surprisingly little is known about the mechanism of gene expression in tick salivary glands.
furthermore, the exact tissue localization of the sulfotransferases in the nymphal and larval stage has not yet been determined; whole homogenates were used of unfed and blood-fed ticks at these life stages.
the related sulfotransferase  isolated from c. elegans catalyzed sulfonation of a variety of phenols at the rate of  <dig> - <dig>  nmol min- <dig> mg- <dig>  but no endogenous substrate was identified  <cit> .
the sulfotransferase  isolated from bombyx mori was found to catalyze sulfonation of 4-nitrocatechol at  <dig>  nmol min- <dig> mg- <dig>  although no endogenous substrate was identified  <cit> .
the cytosolic sulfotransferases from d. melanogaster catalyzed sulfonation of vanillin, 1-naphthol, p-nitrophenol, ecdysone, and dopamine  <cit>  at rates of  <dig> - <dig> μmol min- <dig> mg- <dig>  the published rates of sulfonation by bmst and cest are similar to those measured for ixosc sult  <dig> and  <dig>
the well-characterized human sulfotransferases catalyze sulfonation of a variety of endogenous hormones and neurotransmitters, as well as dietary phenols and environmental contaminants.
in general, the sulfotransferases responsible for sulfonating endogenous substrates have high affinity for these substrates.
human sult1a <dig>  which is also called dopamine sulfotransferase, has a km of  <dig> μm for dopamine, while human sult1a, generally called phenol sulfotransferase, has a km of  <dig> μm for dopamine.
human sult1e <dig>  which is also called estrogen sulfotransferase, has a km of 1- <dig> nm for 17β-estradiol, while human sult1a <dig> and sult2a <dig> can sulfonate 17β-estradiol with km of 1- <dig> μm.
in our earlier findings, dopamine was found to be a good substrate  for the native tick sulfotransferases irrespective of their feeding stage where as octopamine served as a substrate only after feeding  <cit> .
interestingly, in this present study, we found that both dopamine and r,s-octopamine could serve as substrates for purified ixosc sult  <dig>  and sult  <dig>  at a concentration of  <dig> μm.
we chose  <dig> μm as a test concentration because of our detection method's limit of sensitivity.
further studies are needed to explore the physiological relevance of ixosc sult  <dig> and sult  <dig> sulfonation of tick neuroeffectors dopamine and octopamine, and to identify possible additional sulfotransferases in ixodes scapularis.
we find the sulfonation of dopamine and octopamine to be noteworthy because salivary secretion in feeding ticks is under neuronal control.
tick salivary gland secretion is stimulated by dopamine in the neuroeffector junction via dopamine d <dig> receptor activation of adenylate cyclase and an increase in intracellular camp  <cit> .
dopamine also opens a voltage-gated ca2+ channel allowing an influx of extracellular calcium that stimulates a cytosolic phospholipase a <dig>  effecting release of sequestered arachidonic acid in tick salivary glands that is subsequently converted to prostaglandins  <cit> .
prostaglandins are secreted at extremely high  levels into tick saliva for export to the host  <cit>  where they exhibit anti-hemostatic, vasodilatory, immuno-suppressive, and anti-inflammatory activities  <cit> .
thus, dopamine could modulate salivation and/or prostaglandin production in the tick salivary gland.
octopamine acts as a neuromodulator in arthropods with a similar mechanism to dopamine  <cit> .
in insects, octopaminergic neurons modulate a variety of target tissues, which include skeletal muscles, heart and oviduct  <cit> .
however, the exact tissue localization of the sulfotransferases in nymphal and larval blacklegged ticks has not yet been determined; whole homogenates were used of unfed and blood-fed ticks at these life stages.
in contrast to vertebrates, invertebrates use a variety of enzymatic routes to metabolise monoamines.
these routes include sulfonation, n-acetylation, gamma-glutamyl conjugation, sugar conjugation, β-alanyl conjugation, as well as oxidative deamination  <cit> .
catabolism is one of the effective mechanisms for dopamine inactivation.
in humans, this involves multiple pathways that include oxidative deamination by monoamine oxidase , o-methylation by catechol-o-methyltransferase  and conjugation by sulfotransferase  <cit> .
regarding ticks, reports from atkinson et al.
<cit>  confirm the presence of monoamine oxidase  in the homogenates of b. microplus larvae, and mao inhibition potentiated the dopamine effect on fluid secretion from salivary glands of a. hebraeum in vitro  <cit> .
according to in vivo experiments by kaufman and sloley  <cit> , it is clear that mao is not the only means for disposing of biogenic amines in ticks.
octopamine n-acetyltransferase  has been demonstrated in the synganglion of b. microplus  <cit> .
thus, multiple pathways may be available for inactivating monoamines in tick species.
indeed, despite the recent advances in tick neurobiology and the identification of key genes, tick neuroscience lags behind that other invertebrates  <cit> .
ixosc sult  <dig> or sult  <dig> may serve as critical modulators of the prostaglandin synthesis pathway or as modulators of salivary secretion.
because all neuronally released biogenic amines have relatively short half-lives to prevent their action from continuing after the neuronal signal arrests, we thus propose that the two tick sulfotransferases described herein could function by metabolically altering the biological signal for salivary secretion in i. scapularis even though both of these genes were expressed differently.
we are currently following up on these studies to explore their physiological relevance to tick feeding.
CONCLUSIONS
the salivary glands of ticks, in addition to their role in feeding, serve a role in ion and water metabolism.
in a blood-feeding tick, production of saliva is the main mechanism of water excretion, ie.
ticks alternate blood ingestion and salivation.
therefore, interrupted or inappropriate sulfation of neurotransmitter molecules involved in salivation could negatively affect a tick's ability to alternate the cyclic salivation and blood sucking process, potentially inhibiting blood feeding and pathogen transmission.
in this study, we observed that expression of ixosc sult  <dig> is up-regulated, while expression of ixosc sult  <dig> is down-regulated during blood feeding at both the larval and nymphal stages, suggesting that these sulfotransferases have different functions in the salivation/feeding process of blacklegged ticks, even though expressed recombinant proteins sulfonate both dopamine and octopamine.
further experiments, perhaps using sulfotransferase gene knockdown in these ticks may help clarify the significance of our observations.

 ticks are hematophagous arthropods, notorious as vectors of human and animal pathogens. diseases transmitted by ticks are global medical and veterinary public health problems. ixodes scapularis, known commonly as the deer tick or blacklegged tick, is the major vector for lyme disease, babesiosis, and granulocytic anaplasmosis to humans and domestic animals in the united states. ticks and other blood-feeding arthropod vectors manipulate host hemostatic and immune responses by secreting molecules from their multifunctional salivary glands. blood feeding by ticks requires prolonged contact with host tissues and blood, and it has been suggested that the adaptation of ticks to their natural host has resulted in evolution of an appropriate set of salivary components allowing the tick to evade host immunity and prevent coagulation at the feeding site to successfully obtain its blood meal. the blood feeding cycle of larval and nymphal ixodes scapularis typically extends for three to four days, while that of the adult female blacklegged tick lasts approximately six days; during this time the tick alternately secretes salivary fluid into the host and takes up blood from the host. successful blood feeding must require endogenous signalling molecules within the tick to turn on and off salivation and control the release of some secreted salivary proteins into the saliva. one possible strategy for reducing tick-transmitted disease incidence could involve manipulating tick salivary secretion so as to interrupt or shorten the duration of host attachment or blood feeding. accordingly, this research is focussed on elucidating biochemical pathways of two sulfotransferase genes that may be involved in tick salivation and pathogen transmission. sulfotransferases catalyze transfer of a sulfonyl moiety from the universal donor 3'-phosphoadenosine-5'-phosphosulfate to an oxygen or nitrogen acceptor, resulting in production of a sulfate ester. historically, sulfotransferases have been classified by their sub-cellular localization as either cytosolic or membrane bound. in general, cytosolic sulfotransferases accept relatively small molecules as substrates, while the membrane bound sulfotransferases utilize macromolecules. although a number of sulfotransferase genes are registered in various invertebrate genome databases, only a few invertebrate sulfotransferases have been characterized. cytosolic sulfotransferases have been characterized from the nematode caenorhabditis elegans and from the arthropods bombyx mori, spodoptera frugiperda, and drosophila melanogaster. c. elegans contains a glycosaminoglycan membrane bound sulfotransferase with heparan-2-o-sulfotransferase activity. membrane bound tyrosylprotein sulfotransferases have been characterized from drosophila melanogaster and from c. elegans. specifically in salivary glands, tyrosylprotein sulfotransferases have been characterized from human saliva and rat salivary gland tissue. thus, precedent exists for both cytosolic and membrane-localized sulfotransferases in arthropods, and in mammalian salivary glands. sulfotransferase enzymes modulate the activity of many hormones and proteins; in humans and other mammalian species, some are involved in regulating immune responses and blood coagulation. recently, we predicted that dopamine and octopamine could serve as substrates for tick-derived sulfotransferases. dopamine is known to stimulate secretion of tick saliva through a neurochemical mechanism. tick salivary fluid secretion is controlled via a dopamine d receptor and camp dependent protein phosphorylation cascade following salivary gland stimulation by dopamine released from nerve endings. moreover, endogenous dopamine has been identified in salivary glands of the ticks rhipicephalus microplus and amblyomma hebraeum. octopamine also exhibits neuromodulatory functions in arthropods. thus, precedent exists for sulfotransferase enzymes to impact host hemostasis as well as modulate tick salivary or host components which regulate salivation, immune response, or blood coagulation. our recent report showed the three dimensional structural conservation of two novel tick sulfotransferases and its ligand docking. predictions from the modelling were tested and confirmed using native tissue enzyme homogenates from larval and nymphal stage blacklegged ticks. in this present study, we identify the genetic expression of two cytosolic sulfotransferases throughout the tick life cycle and characterize the expressed recombinant proteins. interestingly, we observed differential gene expression between the two sulfotransferases during blood feeding, and that their recombinant enzymes effectively sulfate neurotransmitters dopamine and octopamine. taken together, the observed differential expression of these two sulfotransferase genes and the resulting protein affinity for neurotransmitters involved in tick salivation suggests that ixosc sult and sult may play roles in tick salivation and pathogen transmission processes.  amino acid sequence comparison the sequence alignment of the two ixodes sulfotransferases and related mammalian cytosolic sulfotransferases are shown in figure the sult_motif on figure indicates the residues which make up the cytosolic sulfotransferase amino acid sequence motif regions. thus, the amino acid sequence comparison confirms that there is presence of conserved domains of cytosolic sulfotransferases on both ixosc sult and transcriptional expression of ixosc sult and sult genes and gene products to shed light on the transcriptional control of the two genes during different life stages of i. scapularis, we used real-time quantitative rt-pcr to amplify rna isolated from pooled unfed and blood-fed whole larvae and nymphs, as well as the salivary glands and midguts dissected from both unfed and blood-fed adult stage ticks. usually, the feeding time varied between to hours. expression levels were normalized using the constitutively expressed β-actin transcript as a standard. results showed that the sult was increased during feeding fold in larvae and fold in nymphs. in contrast, sult was decreased during feeding by fold in larvae and fold in nymphs as shown in figure 2a and 2b. furthermore, the difference in transcript abundance for sult and sult were statistically significant, p < for sult and p < for sult during larval stages between unfed and fed, where as p < and p < for sult during nymphal stages between unfed and fed, respectively. briefly, as feeding initiated in larval ticks, sult expression increased whereas sult expression decreased during feeding in both larval and nymphal stages. during the adult tick feeding, there was no significant difference in sult or sult transcript abundance. similar results were observed at the protein level using western blot probed with antibodies raised against expressed and affinity-purified ixosc sult or sult band intensities were quantified using kodak digital science 1d image analysis software and plotted against different tick stages. values are expressed as mean ± sd for three replicated experiments and show significant differences between blood-fed and unfed states, respectively, at both the nymphal and larval stages. similar to the quantitative pcr results, the ixosc sult and protein levels in the adult stage showed no difference on feeding. full length ixosc sult and sult were cloned into the ptrchis topo ta expression vector. the expressed proteins were purified using a his-select ilap column. the purified ixosc sult and sult showed molecular weights of and kda, respectively, and the purified proteins were sequenced using liquid automatic sequencing. the resulting sequences from the expert protein analysis system confirmed that the expressed proteins are from tick sulfotransferase genes. enzymatic characterization of ixosc sult and sult we tested whether expressed purified ixosc sult or sult could sulfonate selected potentially relevant substrates. the rationale for including p-nitrophenol, 17b-estradiol, pregnenolone, and dopamine was that they are prototype substrates for the mammalian sulfotransferases. the rationale for including octopamine and an additional rationale for including dopamine was based on the relevance of these compounds as arthropod neuromodulators and on the previous structure modelling study which predicted these monoamines as potential substrates for the i. scapularis sulfotransferases. arthropod steroids such as ecdysone were not tested as substrates, as the previous structure modelling study indicated that these steroid structures are not complimentary to the binding pocket of the i. scapularis sulfotransferases and should not be substrates. each potential substrate was added to μm pap35s and purified enzyme, and was incubated at 37°c. because of the zwitterionic nature of two of the expected products at acidic ph, we used cellulose thin-layer chromatography under strongly basic conditions for the separation. as shown in figure and quantified in table p-nitrophenol, dopamine, and r,s-octopamine were substrates for both ixosc sult and sult. the enzyme activity of expressed ixosc sult was found to be - nmol min- mg- and ixosc sult to be - nmol min- mg- respectively, when incubated with μm of each potential substrate. product formation in the absence of enzyme was found to be zero. 17β-estradiol and pregnenolone showed no product formation under the conditions tested. because the 35s-labeled products of dopamine, octopamine, and p-nitrophenol migrated with a similar retention factor, their individual formation was confirmed by mass spectral analysis. the sulfated product of dopamine and octopamine, respectively, were positively identified by mass spectral detection under negative ionization conditions. p-nitrophenylsulfate ion was detected in parallel incubations with both ixosc sult and and sult. tlc separation of 35s-labeled product from pap35s after incubation of μm of noted compounds with expressed purified ixosc sult or sult as described in experimental procedures. a substrates were incubated with pap35s and purified ixosc sult or ixosc sult for min. product formation in the absence of enzyme was found to be zero. discussion in this study, we describe the expression of novel ixodes scapularis sulfotransferase genes throughout the blacklegged tick life cycle and different states of blood feeding, and characterize their expressed recombinant proteins. ixosc sult and sult are expressed during every life stage of blacklegged ticks, and interestingly, mrna and protein levels are moderately but significantly changed upon blood-feeding at the nymphal and larval stages. we find it especially interesting that the process of feeding has an opposite effect on relative expression of the ixosc sult and sult during both the larval and nymphal life stages, although no change in expression levels could be detected in the adult stage salivary gland or midgut. thus, these two proteins seem to have different roles in the tick salivation and feeding process. ixosc sult mrna and protein were increased upon feeding from a low basal level at the larval and nymphal stage. in contrast, ixosc sult mrna and protein were decreased upon feeding from a high basal level at the larval and nymphal stage. quantitative rt-pcr and quantification of band intensities by kodak digital analyser confirmed the statistical increase of sult and decrease of sult as well as their gene products during feeding. our recent publication on homology modelling, molecular docking and enzyme kinetic studies of sulfotransferase activity from native tick tissues showed similar findings using specific tick homogenates. it should be noted that these results were obtained from three pools of tissue derived from multiple ticks, and that the blood-fed ticks were sampled after being attached to hosts for - hours. because tick salivation and secretion is a dynamic process throughout the multi-day feeding, experiments are in progress to profile the expression during more exact time periods. while there is evidence for altered expression of multiple genes during tick feeding, surprisingly little is known about the mechanism of gene expression in tick salivary glands. furthermore, the exact tissue localization of the sulfotransferases in the nymphal and larval stage has not yet been determined; whole homogenates were used of unfed and blood-fed ticks at these life stages. the related sulfotransferase isolated from c. elegans catalyzed sulfonation of a variety of phenols at the rate of - nmol min- mg- but no endogenous substrate was identified. the sulfotransferase isolated from bombyx mori was found to catalyze sulfonation of 4-nitrocatechol at nmol min- mg- although no endogenous substrate was identified. the cytosolic sulfotransferases from d. melanogaster catalyzed sulfonation of vanillin, 1-naphthol, p-nitrophenol, ecdysone, and dopamine at rates of - μmol min- mg- the published rates of sulfonation by bmst and cest are similar to those measured for ixosc sult and the well-characterized human sulfotransferases catalyze sulfonation of a variety of endogenous hormones and neurotransmitters, as well as dietary phenols and environmental contaminants. in general, the sulfotransferases responsible for sulfonating endogenous substrates have high affinity for these substrates. human sult1a which is also called dopamine sulfotransferase, has a km of μm for dopamine, while human sult1a, generally called phenol sulfotransferase, has a km of μm for dopamine. human sult1e which is also called estrogen sulfotransferase, has a km of 1- nm for 17β-estradiol, while human sult1a and sult2a can sulfonate 17β-estradiol with km of 1- μm. in our earlier findings, dopamine was found to be a good substrate for the native tick sulfotransferases irrespective of their feeding stage where as octopamine served as a substrate only after feeding. interestingly, in this present study, we found that both dopamine and r,s-octopamine could serve as substrates for purified ixosc sult and sult at a concentration of μm. we chose μm as a test concentration because of our detection method's limit of sensitivity. further studies are needed to explore the physiological relevance of ixosc sult and sult sulfonation of tick neuroeffectors dopamine and octopamine, and to identify possible additional sulfotransferases in ixodes scapularis. we find the sulfonation of dopamine and octopamine to be noteworthy because salivary secretion in feeding ticks is under neuronal control. tick salivary gland secretion is stimulated by dopamine in the neuroeffector junction via dopamine d receptor activation of adenylate cyclase and an increase in intracellular camp. dopamine also opens a voltage-gated ca2+ channel allowing an influx of extracellular calcium that stimulates a cytosolic phospholipase a effecting release of sequestered arachidonic acid in tick salivary glands that is subsequently converted to prostaglandins. prostaglandins are secreted at extremely high levels into tick saliva for export to the host where they exhibit anti-hemostatic, vasodilatory, immuno-suppressive, and anti-inflammatory activities. thus, dopamine could modulate salivation and/or prostaglandin production in the tick salivary gland. octopamine acts as a neuromodulator in arthropods with a similar mechanism to dopamine. in insects, octopaminergic neurons modulate a variety of target tissues, which include skeletal muscles, heart and oviduct. however, the exact tissue localization of the sulfotransferases in nymphal and larval blacklegged ticks has not yet been determined; whole homogenates were used of unfed and blood-fed ticks at these life stages. in contrast to vertebrates, invertebrates use a variety of enzymatic routes to metabolise monoamines. these routes include sulfonation, n-acetylation, gamma-glutamyl conjugation, sugar conjugation, β-alanyl conjugation, as well as oxidative deamination. catabolism is one of the effective mechanisms for dopamine inactivation. in humans, this involves multiple pathways that include oxidative deamination by monoamine oxidase, o-methylation by catechol-o-methyltransferase and conjugation by sulfotransferase. regarding ticks, reports from atkinson et al. confirm the presence of monoamine oxidase in the homogenates of b. microplus larvae, and mao inhibition potentiated the dopamine effect on fluid secretion from salivary glands of a. hebraeum in vitro. according to in vivo experiments by kaufman and sloley, it is clear that mao is not the only means for disposing of biogenic amines in ticks. octopamine n-acetyltransferase has been demonstrated in the synganglion of b. microplus. thus, multiple pathways may be available for inactivating monoamines in tick species. indeed, despite the recent advances in tick neurobiology and the identification of key genes, tick neuroscience lags behind that other invertebrates. ixosc sult or sult may serve as critical modulators of the prostaglandin synthesis pathway or as modulators of salivary secretion. because all neuronally released biogenic amines have relatively short half-lives to prevent their action from continuing after the neuronal signal arrests, we thus propose that the two tick sulfotransferases described herein could function by metabolically altering the biological signal for salivary secretion in i. scapularis even though both of these genes were expressed differently. we are currently following up on these studies to explore their physiological relevance to tick feeding.  the salivary glands of ticks, in addition to their role in feeding, serve a role in ion and water metabolism. in a blood-feeding tick, production of saliva is the main mechanism of water excretion, ie. ticks alternate blood ingestion and salivation. therefore, interrupted or inappropriate sulfation of neurotransmitter molecules involved in salivation could negatively affect a tick's ability to alternate the cyclic salivation and blood sucking process, potentially inhibiting blood feeding and pathogen transmission. in this study, we observed that expression of ixosc sult is up-regulated, while expression of ixosc sult is down-regulated during blood feeding at both the larval and nymphal stages, suggesting that these sulfotransferases have different functions in the salivation/feeding process of blacklegged ticks, even though expressed recombinant proteins sulfonate both dopamine and octopamine. further experiments, perhaps using sulfotransferase gene knockdown in these ticks may help clarify the significance of our observations. 
interestingly, the resultant recombinant proteins showed sulfotransferase activity against both neurotransmitters dopamine and octopamine. the sulfotransferase genes were cloned and expressed in a bacterial expression system, and purified recombinant proteins ixosc sult and showed sulfotransferase activity against neurotransmitters dopamine and octopamine as well as the common sulfotransferase substrate p-nitrophenol. in this current study, we characterize the genetic expression of these two cytosolic sulfotransferases throughout the tick life cycle as well as the enzymatic properties of the corresponding recombinant proteins. the two sulfotransferase genes were coded as ixosc sult & and corresponding proteins were referred as ixosc sult and using gene-specific primers, the sulfotransferase transcripts were detected throughout the blacklegged tick life cycle, including eggs, larvae, nymphs, adult salivary glands and adult midgut. thus, dopamine- or octopamine-sulfonation may be involved in altering the biological signal for salivary secretion in i. scapularis. ixodes scapularis, commonly known as the blacklegged or deer tick, is the main vector of lyme disease in the united states. recent progress in transcriptome research has uncovered hundreds of different proteins expressed in the salivary glands of hard ticks, the majority of which have no known function, and include many novel protein families. collectively, these results suggest that a function of ixosc sult and sult in ixodid tick salivary glands may include inactivation of the salivation signal via sulfonation of dopamine or octopamine. quantitative pcr results confirm that ixosc sult was statistically increased upon blood feeding while ixosc sult was decreased. notably, the mrna and protein levels were altered upon feeding during both the larval and nymphal life stages. we recently identified transcripts coding for two putative cytosolic sulfotransferases in these ticks which recognized phenolic monoamines as their substrates.
 ixodes scapularis, commonly known as the blacklegged or deer tick, is the main vector of lyme disease in the united states. recent progress in transcriptome research has uncovered hundreds of different proteins expressed in the salivary glands of hard ticks, the majority of which have no known function, and include many novel protein families. we recently identified transcripts coding for two putative cytosolic sulfotransferases in these ticks which recognized phenolic monoamines as their substrates. in this current study, we characterize the genetic expression of these two cytosolic sulfotransferases throughout the tick life cycle as well as the enzymatic properties of the corresponding recombinant proteins. interestingly, the resultant recombinant proteins showed sulfotransferase activity against both neurotransmitters dopamine and octopamine.  the two sulfotransferase genes were coded as ixosc sult & and corresponding proteins were referred as ixosc sult and using gene-specific primers, the sulfotransferase transcripts were detected throughout the blacklegged tick life cycle, including eggs, larvae, nymphs, adult salivary glands and adult midgut. notably, the mrna and protein levels were altered upon feeding during both the larval and nymphal life stages. quantitative pcr results confirm that ixosc sult was statistically increased upon blood feeding while ixosc sult was decreased. this altered expression led us to further characterize the function of these proteins in the ixodid tick. the sulfotransferase genes were cloned and expressed in a bacterial expression system, and purified recombinant proteins ixosc sult and showed sulfotransferase activity against neurotransmitters dopamine and octopamine as well as the common sulfotransferase substrate p-nitrophenol. thus, dopamine- or octopamine-sulfonation may be involved in altering the biological signal for salivary secretion in i. scapularis.  collectively, these results suggest that a function of ixosc sult and sult in ixodid tick salivary glands may include inactivation of the salivation signal via sulfonation of dopamine or octopamine. 