BACKGROUND
motile cilia are microscopic membrane-bound extensions of certain cells that are vital for the survival and reproduction of many eukaryotes.
by beating in a regular pattern these evolutionarily-conserved organelles exert mechanical force; they thus play important roles in motility of small organisms and facilitate fluid movement across epithelial surfaces in complex multicellular animals.
in addition to their role in fluid movement, motile cilia have recently been found to possess sensory functions in mammals, a feature previously thought to be restricted to non-motile 'primary' cilia  <cit> .
given that motile cilia are essential to physiology it is not surprising that defects in these organelles cause multiple human disorders  <cit> .
such ciliopathies include primary ciliary dyskinesia resulting in an inability to clear mucous and debris from airways  <cit> , hydrocephalus caused by abnormal spinal fluid movement in the ventricles of the brain  <cit> , and situs inversus  a consequence of altered nodal flow during embryogenesis  <cit> .
motile cilia  are also essential for the completion of the life cycles of various parasites of humans and animals.
for example, they power locomotion of schistosome larvae  enabling host-finding and thus infection of the snail intermediate host  <cit> , and are required for migration of trypanosomes between gut and salivary glands in the tsetse fly vector  <cit> , and for viability of the bloodstream trypanosome form  <cit> .
motile cilia  are composed of nine microtubular doublets and, usually, two central mictrotubular singlets, comprising the axoneme; dynein arms and radial spokes associated with the axoneme generate and control axonemal bending and thus force generation.
the regulation of ciliary beating has been the focus of much research  <cit> , and proteomic studies including those on the model organism chlamydomonas reinhardtii, have aimed to describe the repertoire of proteins present within eukaryotic cilia and flagella, or their component fractions  <cit> .
the proteomic analysis of chlamydomonas flagella revealed over  <dig> putative signal transduction proteins including kinases and phosphatases  <cit> , some of which might be anchored to the axoneme  <cit> , highlighting the importance of signalling and reversible protein phosphorylation in the function of motile cilia and flagella.
this is further supported by the recent identification in this organelle of  <dig> flagella phosphoproteins with  <dig> in vivo phosphorylation sites  <cit> .
despite these advances, our understanding of kinase-mediated cell signalling mechanisms regulating ciliary motion is still rudimentary, being largely restricted to the roles of the camp-dependent protein kinase, protein kinase a   <cit> , and protein kinase c   <cit> .
the surface of the schistosome miracidium is almost entirely covered with numerous motile locomotory cilia which, in schistosoma mansoni, emerge from  <dig> ciliated epidermal plates arranged in tiers  <cit> .
the s. mansoni miracidium is fully developed in the egg when it is released in the faeces of the infected definitive  host; upon water contact the miracidium hatches and swims rapidly to locate a suitable intermediate freshwater snail-host.
miracidia movements respond rapidly to various environmental cues such as the presence of host-snail components  <cit>  or salinity  <cit> , suggesting that these motile cilia might possess sensory functions as is the case with paramecium .
while studying kinase-mediated cell signalling in s. mansoni during miracidia development we observed an unexpected event, namely that activation of p <dig> mitogen-activated protein kinase  attenuated miracidial swimming.
this kinase, an orthologue of the yeast hog kinase, participates in signalling cascades that regulate transcriptional responses to stress  <cit>  as well as having other non-transcription factor targets such as cytosolic phospholipase a <dig>  <cit> .
here we report findings that support a role for p <dig> mapk in the regulation of ciliary motion of the multicellular eukaryote s. mansoni.
RESULTS
characterization of s. mansoni p <dig> mapk
as with other mapks, p <dig> mapk has been highly conserved during metazoan evolution  <cit> .
recently, the draft genomes for s. mansoni and schistosoma japonicum were published  <cit>  allowing s. mansoni p <dig> mapk gene candidates to be identified and further assessed for similarity to p <dig> mapks from other organisms, including closely related schistosomes .
for s. mansoni only a single putative p <dig> mapk was identified and only partial cdna reads were found; moreover, only one p <dig> mapk was found in s. japonicum .
thus, in contrast to d. melanogaster and human which possess two and four p <dig> mapks respectively  <cit> , it seems that schistosomes may possess one p <dig> mapk orthologue only.
based on the partial sequence data  spanning  <dig> amino acids, and supported by more complete data from s. japonicum, the identified s. mansoni p <dig> mapk was most similar to p38α mapk  of humans, the most evolutionarily-conserved p <dig> mapk  <cit> .
in addition, there exist a number of putative exons on two separate scaffolds  additional to the  <dig> amino acid fragment that, when translated, also match closely with the s. japonicum sequence; thus, we assume given the phylogenetic proximity between the two species that s. mansoni p <dig> mapk is very similar to s. japonicum p <dig> mapk.
as for all other p <dig> mapks in the p <dig> subfamily, the dual phosphorylation site in the s. mansoni p <dig> mapk activation loop reads thr-gly-tyr .
the substrate binding site ala-thr-arg-trp  is also conserved, as is the kinase interaction motif  docking site  which binds linear kim sequences present in substrates and mapk phosphatases  <cit> .
pair wise comparisons of the s. mansoni p <dig> mapk fragment with corresponding sequences for other organisms revealed ~69-70% similarity with human, drosophila melanogaster, caenorhabditis elegans, or danio rerio, and  <dig> % with s. japonicum.
anti-phospho p <dig> mapk monoclonal antibodies were used in an attempt to detect phosphorylated p <dig> mapk in s. mansoni.
these antibodies, that bind p <dig> mapk only when dually phosphorylated on thr/tyr of the tgy motif, have been used to detect phosphorylated p <dig> mapk in many multicellular eukaryotes including c. elegans  <cit>  and d. melanogaster  <cit> .
because phosphorylation on these residues results in activation of the enzyme, immunoreactivity directly correlates with p <dig> mapk activity.
the p <dig> mapk amino acid sequence surrounding the tgy motif to which these antibodies are raised is highly-conserved between both s. mansoni and s. japonicum, and between s. mansoni and c. elegans, d. melanogaster, d. rerio and human .
western blotting of adult worm homogenates revealed that anti-phospho p <dig> mapk antibodies recognized a single protein band with apparent molecular weight of approximately  <dig> kda, essentially co-migrating with phosphorylated p <dig> mapk from human astrocytoma  cells .
in marked contrast to adult worms, freshly-hatched swimming miracidia did not possess detectable levels of phosphorylated p <dig> mapk .
a second antibody  that detects p <dig> mapk in vertebrates irrespective of phosphorylation state did not react with the s. mansoni protein precluding its use in this study.
a p <dig> mapk immunoprecipitation kinase assay kit was next employed to confirm that the s. mansoni protein recognized by the anti-phospho p <dig> mapk antibodies possessed p <dig> mapk activity.
immunoprecipitates from adult worm homogenates phosphorylated the p <dig> mapk substrate activating transcription factor  <dig>  .
the anti-inflammatory pyridinylimidazole compounds  inhibit p <dig> mapk with both activity of the phosphorylated enzyme and its autophosphorylation affected  <cit> .
these inhibitors compete with atp at the atp binding site and exhibit no, or very weak inhibitory activity toward the closely related mapks, c-jun n-terminal kinase  and extracellular signal-regulated kinase   <cit> .
sb  <dig> inhibits both p38α and p38β mapk and the interactions between the inhibitor and human kinases have been mapped using crystallography and amino acid substitution experiments  <cit> .
the crucial residues thought to be involved in this interaction are shown in figure 1; although falling outside the s. mansoni sequence fragment, these residues are conserved in s. japonicum p <dig> mapk.
we thus postulated that sb  <dig> would inhibit s. mansoni p <dig> mapk.
pre-incubation of immunoprecipitates with sb  <dig> prior to kinase assay reduced atf- <dig> phosphorylation considerably when compared to immunoprecipitates incubated in dimethyl sulfoxide  vehicle alone; inhibition appeared dose dependent with  <dig> μm sb  <dig> almost completely attenuating atf- <dig> phosphorylation .
thus the antibody recognizes an s. mansoni protein with p <dig> mapk-like activity that can be inhibited by the p <dig> mapk inhibitor sb  <dig>
anisomycin is a known potent stimulator of p <dig> mapk phosphorylation, and therefore activation, so we considered that this compound might activate p <dig> mapk in freshly-hatched swimming miracidia that lacked the phosphorylated  form of the enzyme .
western blotting revealed that treatment of these miracidia with  <dig> μm anisomycin for  <dig> min induced considerable p <dig> mapk phosphorylation when compared to controls, which appeared to increase after  <dig> min and was sustained over  <dig> min .
the results of the above biochemical experiments, coupled with the bioinformatic analysis, are commensurate with the s. mansoni immunoreactive protein being a p <dig> mapk orthologue.
activated p <dig> mapk is associated with s. mansoni cilia
to determine the localization of activated p <dig> mapk within s. mansoni miracidia, anti-phospho p <dig> mapk antibodies and confocal laser scanning microscopy were used.
z-axis projections viewed in maximum pixel brightness mode revealed that freshly-hatched swimming miracidia possessed low levels of phosphorylated p <dig> mapk, which is in agreement with western blotting analysis .
however where activation was evident, it appeared to be associated with the region occupied by the cilia .
miracidia incubated with secondary antibodies alone possessed only background fluorescence .
when freshly-hatched swimming miracidia were incubated with  <dig> μm anisomycin for  <dig> min, a striking increase in p <dig> mapk activation was observed  whereas dmso exposed  miracidia appeared similar to untreated miracidia .
analysis of serial optical z-sections revealed activation largely at, or adjacent to, the ciliated surface of the parasite .
fine z-sectioning and optical zooming revealed that activated p <dig> mapk was associated with the shaft of the cilia , further demonstrated through co-localization using anti-acetylated tubulin antibodies that permit visualization of these structures .
immunoreactivity was present up to 6- <dig> μm from the tegument surface which correlates with the length  of the cilia reported from transmission electron microscopy studies  <cit> .
staining for activated p <dig> mapk was also observed in the tegument ; here the enzyme could either be associated with the base of the cilia, the microvilli  that are known to exist among the cilia, the rootlet  that is thought to provide support  <cit> , or other structures.
the semi-spherical terebratorium  lacks cilia  but possess filopodia-like sensory endings  <cit>  thought to play a role in sensing the intermediate snail host.
although staining was observed at the terebratorium, it was noticeably less than that observed in the adjacent ciliated plates .
while observing miracidia by immunofluorescence microscopy a solitary egg was found on the microscope slide, this egg had ruptured to allow hatching but fortunately still contained the miracidium which stained successfully with antibodies.
strikingly, and in contrast to swimming miracidia, the hatching miracidium possessed considerable p <dig> mapk activity that via z-section analysis was found to be cilia-associated.
experiments were then designed in an attempt to re-capture this event and on several occasions miracidia were observed within ruptured eggs; such miracidia contained cilia-associated p <dig> mapk activity considerably greater than that of freshly-hatched swimming miracidia .
although various kinases including pka, casein kinase, adenylate kinase, cgmp-dependent protein kinase and a putative mapk have been identified in proteomic screens of cilia/flagella e.g.
<cit> , to our knowledge p <dig> mapk has not been detected by such methods.
p <dig> mapk has however recently been localized to the post-acrosomal region and upper flagellum mid-piece of human sperm by fluorescence microscopy  <cit> .
although sperm tails are classified as flagella, their regulation differs from motile cilia in some aspects; sperm also possess a number of unique accessory structures  <cit>  making them somewhat distinct from other motile cilia.
activation of p <dig> mapk correlates with attenuation of cilia beat and swim velocity
because p <dig> mapk was active in stationary miracidia within eggs but inactive in swimming miracidia, and because p <dig> mapk localized to cilia, we next explored whether pharmacological activation or inhibition of p <dig> mapk phenotypically affected ciliary function by performing swim velocity analyses.
miracidia were exposed to anisomycin or sb  <dig> and their swim behaviour recorded by digital video microscopy .
determination of swim velocities revealed that freshly-hatched miracidia swam at speeds between  <dig>  and  <dig>  mm/s over  <dig> min ; dmso treatment did not affect swim velocities over this duration .
however, anisomycin  significantly attenuated miracidia swim velocity over time when compared to untreated  controls  ; after  <dig> min and  <dig> min mean swim velocity was reduced by 25% to  <dig>  mm/s , and by 80% to  <dig>  mm/s , respectively.
after  <dig> min anisomycin treatment swimming essentially ceased.
on the other hand, sb  <dig>  significantly accelerated miracidia swim velocity , particularly after  <dig> and  <dig> min treatment when it increased ~ <dig> % .
importantly, following  <dig> min anisomycin treatment, when spring water  or sb  <dig>  were added, the miracidia started swimming displaying swim velocities of between  <dig>  and  <dig>  mm/s after  <dig> min .
that the effects of anisomycin could be reversed demonstrates that cessation of swimming was not due to larval death.
thus, the contrasting effects of sb  <dig> and anisomycin on swim velocity and the finding that p <dig> mapk localizes to the cilia are consistent with p <dig> mapk playing a role in ciliary beat and thus miracidia swimming.
anisomycin is thought to indirectly activate p <dig> mapk via cdc42/rac, mapkkks, mapkk <dig>  and mkk <dig> which ultimately phosphorylate p <dig> mapk  <cit> .
importantly, orthologues of these pathway components have been identified in s. mansoni  <cit>  suggesting that the mechanisms by which anisomycin exerts its effect on p <dig> mapk are conserved between schistosomes and higher eukaryotes.
p <dig> mapk phosphatases have also been identified in s. mansoni  <cit> , allowing rapid dephosphorylation of p <dig> mapk when required.
although aspects of sperm flagella make them distinct from other motile cilia it is interesting that p <dig> mapk inhibition by sb  <dig> was recently shown to stimulate forward and hyperactivated motility of human sperm  <cit> .
the mechanism by which p <dig> mapk controls ciliary beat is not known.
active p <dig> mapk could attenuate ciliary beat either by direct or indirect interactions involving phosphorylation of axonemal components or components within a ciliary signal transduction cascade, respectively.
studies with c. reinhardtii have demonstrated that phosphorylation and dephosphorylation control flagella motility  <cit>  and have highlighted the complex nature of protein phosphorylation in this organelle  <cit> .
many potential kinase substrates exist that co-ordinate ciliary motility including the dyneins  <cit>  and central pair kinesin klp <dig>  <cit> , but further characterisation of kinase substrates within cilia is needed.
only a few other kinases have been reported to regulate cilia beat.
pka has been shown to attach to paramecium ciliary axonemes, and strong evidence exists for pka playing a positive role in paramecium swimming and in controlling ciliary beat frequency of mammalian cilia  <cit> .
interestingly, pka has also been implicated in regulating the ciliary motion of s. mansoni miracidia  <cit> .
while cgmp-dependent protein kinase  has also been shown to positively regulate ciliary beat frequency  <cit> , like for p <dig> mapk, pkc has been implicated in slowing ciliary beat  <cit> .
clearly, more research is needed into the influence of kinase activities on ciliary movement.
for example, erk1/ <dig> has recently been found to bind radial spoke protein  <dig> in mammals and regulate its interaction with pka  <cit> ; however, the extent to which erk actually influences motility via this interaction warrants investigation.
activation of p <dig> mapk accelerates loss of cilia during larval transformation
when miracidia penetrate a suitable intermediate snail host they shed their ciliated plates and develop into the next larval stage, the mother sporocyst.
this transformation can also be performed in vitro  <cit>  during which miracidia stop swimming before the plates are released.
as p <dig> mapk activation attenuates miracidia swim speed whereas inhibition accelerates it we explored the effects of anisomycin and sb  <dig> on deciliation and thus swimming during miracidium-to-mother sporocyst transformation in vitro.
dmso did not affect the deciliation rate when compared to chernin's balanced salt solution  controls and none of the treatments affected the survival of the developing larvae .
anisomycin accelerated the shedding of ciliary plates considerably ; after only  <dig> h transformation  <dig> % of parasites had stopped swimming having shed at least some cilia in contrast to only  <dig> % of parasites in the cbss control group .
at this time point  <dig> % of anisomycin-treated larvae had shed all their ciliated plates compared to none in cbss alone .
this effect of anisomycin persisted throughout larval transformation .
although the effects of sb  <dig> were somewhat less marked, at  <dig> h,  <dig> h and  <dig> h significantly more miracidia were observed swimming  than were present in the cbss control group .
thus, p <dig> mapk activation appears to accelerate the initial rate of s. mansoni miracidium transformation by attenuating cilia-mediated swimming behaviour leading to early release of ciliated plates.
CONCLUSIONS
here biochemical, innunohistochemical and functional data are presented that are consistent with p <dig> mapk playing an important part in the regulation of ciliary beat and thus swimming behaviour of the multicellular eukaryote, s. mansoni.
the marked difference in p <dig> mapk activation between un-hatched or stationary miracidia and actively swimming miracidia is striking.
localization of active p <dig> mapk to both the cilium shaft and the tegument of stationary miracidia implies that p <dig> mapk might play multiple parts in co-ordinating swim behaviour, including sensory roles as described/hypothesized for motile cilia in other organisms including parasites  <cit> .
given the conservation of both signalling processes and structure/function of motile cilia, we hypothesize that p <dig> mapk might regulate ciliary beat frequency in a variety of metazoans.
thus our findings could have implications for studies into motility of other important multicellular eukaryotes including parasites of humans, and for research into various human ciliopathies.

 motile cilia are microscopic membrane-bound extensions of certain cells that are vital for the survival and reproduction of many eukaryotes. by beating in a regular pattern these evolutionarily-conserved organelles exert mechanical force; they thus play important roles in motility of small organisms and facilitate fluid movement across epithelial surfaces in complex multicellular animals. in addition to their role in fluid movement, motile cilia have recently been found to possess sensory functions in mammals, a feature previously thought to be restricted to non-motile'primary' cilia. given that motile cilia are essential to physiology it is not surprising that defects in these organelles cause multiple human disorders. such ciliopathies include primary ciliary dyskinesia resulting in an inability to clear mucous and debris from airways, hydrocephalus caused by abnormal spinal fluid movement in the ventricles of the brain, and situs inversus a consequence of altered nodal flow during embryogenesis. motile cilia are also essential for the completion of the life cycles of various parasites of humans and animals. for example, they power locomotion of schistosome larvae enabling host-finding and thus infection of the snail intermediate host, and are required for migration of trypanosomes between gut and salivary glands in the tsetse fly vector, and for viability of the bloodstream trypanosome form. motile cilia are composed of nine microtubular doublets and, usually, two central mictrotubular singlets, comprising the axoneme; dynein arms and radial spokes associated with the axoneme generate and control axonemal bending and thus force generation. the regulation of ciliary beating has been the focus of much research, and proteomic studies including those on the model organism chlamydomonas reinhardtii, have aimed to describe the repertoire of proteins present within eukaryotic cilia and flagella, or their component fractions. the proteomic analysis of chlamydomonas flagella revealed over putative signal transduction proteins including kinases and phosphatases, some of which might be anchored to the axoneme, highlighting the importance of signalling and reversible protein phosphorylation in the function of motile cilia and flagella. this is further supported by the recent identification in this organelle of flagella phosphoproteins with in vivo phosphorylation sites. despite these advances, our understanding of kinase-mediated cell signalling mechanisms regulating ciliary motion is still rudimentary, being largely restricted to the roles of the camp-dependent protein kinase, protein kinase a, and protein kinase c. the surface of the schistosome miracidium is almost entirely covered with numerous motile locomotory cilia which, in schistosoma mansoni, emerge from ciliated epidermal plates arranged in tiers. the s. mansoni miracidium is fully developed in the egg when it is released in the faeces of the infected definitive host; upon water contact the miracidium hatches and swims rapidly to locate a suitable intermediate freshwater snail-host. miracidia movements respond rapidly to various environmental cues such as the presence of host-snail components or salinity, suggesting that these motile cilia might possess sensory functions as is the case with paramecium. while studying kinase-mediated cell signalling in s. mansoni during miracidia development we observed an unexpected event, namely that activation of p mitogen-activated protein kinase attenuated miracidial swimming. this kinase, an orthologue of the yeast hog kinase, participates in signalling cascades that regulate transcriptional responses to stress as well as having other non-transcription factor targets such as cytosolic phospholipase a. here we report findings that support a role for p mapk in the regulation of ciliary motion of the multicellular eukaryote s. mansoni.  characterization of s. mansoni p mapk as with other mapks, p mapk has been highly conserved during metazoan evolution. recently, the draft genomes for s. mansoni and schistosoma japonicum were published allowing s. mansoni p mapk gene candidates to be identified and further assessed for similarity to p mapks from other organisms, including closely related schistosomes. for s. mansoni only a single putative p mapk was identified and only partial cdna reads were found; moreover, only one p mapk was found in s. japonicum. thus, in contrast to d. melanogaster and human which possess two and four p mapks respectively, it seems that schistosomes may possess one p mapk orthologue only. based on the partial sequence data spanning amino acids, and supported by more complete data from s. japonicum, the identified s. mansoni p mapk was most similar to p38α mapk of humans, the most evolutionarily-conserved p mapk. in addition, there exist a number of putative exons on two separate scaffolds additional to the amino acid fragment that, when translated, also match closely with the s. japonicum sequence; thus, we assume given the phylogenetic proximity between the two species that s. mansoni p mapk is very similar to s. japonicum p mapk. as for all other p mapks in the p subfamily, the dual phosphorylation site in the s. mansoni p mapk activation loop reads thr-gly-tyr. the substrate binding site ala-thr-arg-trp is also conserved, as is the kinase interaction motif docking site which binds linear kim sequences present in substrates and mapk phosphatases. pair wise comparisons of the s. mansoni p mapk fragment with corresponding sequences for other organisms revealed ~69-70% similarity with human, drosophila melanogaster, caenorhabditis elegans, or danio rerio, and % with s. japonicum. anti-phospho p mapk monoclonal antibodies were used in an attempt to detect phosphorylated p mapk in s. mansoni. these antibodies, that bind p mapk only when dually phosphorylated on thr/tyr of the tgy motif, have been used to detect phosphorylated p mapk in many multicellular eukaryotes including c. elegans and d. melanogaster. because phosphorylation on these residues results in activation of the enzyme, immunoreactivity directly correlates with p mapk activity. the p mapk amino acid sequence surrounding the tgy motif to which these antibodies are raised is highly-conserved between both s. mansoni and s. japonicum, and between s. mansoni and c. elegans, d. melanogaster, d. rerio and human. western blotting of adult worm homogenates revealed that anti-phospho p mapk antibodies recognized a single protein band with apparent molecular weight of approximately kda, essentially co-migrating with phosphorylated p mapk from human astrocytoma cells. in marked contrast to adult worms, freshly-hatched swimming miracidia did not possess detectable levels of phosphorylated p mapk. a second antibody that detects p mapk in vertebrates irrespective of phosphorylation state did not react with the s. mansoni protein precluding its use in this study. a p mapk immunoprecipitation kinase assay kit was next employed to confirm that the s. mansoni protein recognized by the anti-phospho p mapk antibodies possessed p mapk activity. immunoprecipitates from adult worm homogenates phosphorylated the p mapk substrate activating transcription factor. the anti-inflammatory pyridinylimidazole compounds inhibit p mapk with both activity of the phosphorylated enzyme and its autophosphorylation affected. these inhibitors compete with atp at the atp binding site and exhibit no, or very weak inhibitory activity toward the closely related mapks, c-jun n-terminal kinase and extracellular signal-regulated kinase. sb inhibits both p38α and p38β mapk and the interactions between the inhibitor and human kinases have been mapped using crystallography and amino acid substitution experiments. the crucial residues thought to be involved in this interaction are shown in figure 1; although falling outside the s. mansoni sequence fragment, these residues are conserved in s. japonicum p mapk. we thus postulated that sb would inhibit s. mansoni p mapk. pre-incubation of immunoprecipitates with sb prior to kinase assay reduced atf- phosphorylation considerably when compared to immunoprecipitates incubated in dimethyl sulfoxide vehicle alone; inhibition appeared dose dependent with μm sb almost completely attenuating atf- phosphorylation. thus the antibody recognizes an s. mansoni protein with p mapk-like activity that can be inhibited by the p mapk inhibitor sb anisomycin is a known potent stimulator of p mapk phosphorylation, and therefore activation, so we considered that this compound might activate p mapk in freshly-hatched swimming miracidia that lacked the phosphorylated form of the enzyme. western blotting revealed that treatment of these miracidia with μm anisomycin for min induced considerable p mapk phosphorylation when compared to controls, which appeared to increase after min and was sustained over min. the results of the above biochemical experiments, coupled with the bioinformatic analysis, are commensurate with the s. mansoni immunoreactive protein being a p mapk orthologue. activated p mapk is associated with s. mansoni cilia to determine the localization of activated p mapk within s. mansoni miracidia, anti-phospho p mapk antibodies and confocal laser scanning microscopy were used. z-axis projections viewed in maximum pixel brightness mode revealed that freshly-hatched swimming miracidia possessed low levels of phosphorylated p mapk, which is in agreement with western blotting analysis. however where activation was evident, it appeared to be associated with the region occupied by the cilia. miracidia incubated with secondary antibodies alone possessed only background fluorescence. when freshly-hatched swimming miracidia were incubated with μm anisomycin for min, a striking increase in p mapk activation was observed whereas dmso exposed miracidia appeared similar to untreated miracidia. analysis of serial optical z-sections revealed activation largely at, or adjacent to, the ciliated surface of the parasite. fine z-sectioning and optical zooming revealed that activated p mapk was associated with the shaft of the cilia, further demonstrated through co-localization using anti-acetylated tubulin antibodies that permit visualization of these structures. immunoreactivity was present up to 6- μm from the tegument surface which correlates with the length of the cilia reported from transmission electron microscopy studies. staining for activated p mapk was also observed in the tegument; here the enzyme could either be associated with the base of the cilia, the microvilli that are known to exist among the cilia, the rootlet that is thought to provide support, or other structures. the semi-spherical terebratorium lacks cilia but possess filopodia-like sensory endings thought to play a role in sensing the intermediate snail host. although staining was observed at the terebratorium, it was noticeably less than that observed in the adjacent ciliated plates. while observing miracidia by immunofluorescence microscopy a solitary egg was found on the microscope slide, this egg had ruptured to allow hatching but fortunately still contained the miracidium which stained successfully with antibodies. strikingly, and in contrast to swimming miracidia, the hatching miracidium possessed considerable p mapk activity that via z-section analysis was found to be cilia-associated. experiments were then designed in an attempt to re-capture this event and on several occasions miracidia were observed within ruptured eggs; such miracidia contained cilia-associated p mapk activity considerably greater than that of freshly-hatched swimming miracidia. although various kinases including pka, casein kinase, adenylate kinase, cgmp-dependent protein kinase and a putative mapk have been identified in proteomic screens of cilia/flagella e.g., to our knowledge p mapk has not been detected by such methods. p mapk has however recently been localized to the post-acrosomal region and upper flagellum mid-piece of human sperm by fluorescence microscopy. although sperm tails are classified as flagella, their regulation differs from motile cilia in some aspects; sperm also possess a number of unique accessory structures making them somewhat distinct from other motile cilia. activation of p mapk correlates with attenuation of cilia beat and swim velocity because p mapk was active in stationary miracidia within eggs but inactive in swimming miracidia, and because p mapk localized to cilia, we next explored whether pharmacological activation or inhibition of p mapk phenotypically affected ciliary function by performing swim velocity analyses. miracidia were exposed to anisomycin or sb and their swim behaviour recorded by digital video microscopy. determination of swim velocities revealed that freshly-hatched miracidia swam at speeds between and mm/s over min; dmso treatment did not affect swim velocities over this duration. however, anisomycin significantly attenuated miracidia swim velocity over time when compared to untreated controls; after min and min mean swim velocity was reduced by 25% to mm/s, and by 80% to mm/s, respectively. after min anisomycin treatment swimming essentially ceased. on the other hand, sb significantly accelerated miracidia swim velocity, particularly after and min treatment when it increased ~ %. importantly, following min anisomycin treatment, when spring water or sb were added, the miracidia started swimming displaying swim velocities of between and mm/s after min. that the effects of anisomycin could be reversed demonstrates that cessation of swimming was not due to larval death. thus, the contrasting effects of sb and anisomycin on swim velocity and the finding that p mapk localizes to the cilia are consistent with p mapk playing a role in ciliary beat and thus miracidia swimming. anisomycin is thought to indirectly activate p mapk via cdc42/rac, mapkkks, mapkk and mkk which ultimately phosphorylate p mapk. importantly, orthologues of these pathway components have been identified in s. mansoni suggesting that the mechanisms by which anisomycin exerts its effect on p mapk are conserved between schistosomes and higher eukaryotes. p mapk phosphatases have also been identified in s. mansoni, allowing rapid dephosphorylation of p mapk when required. although aspects of sperm flagella make them distinct from other motile cilia it is interesting that p mapk inhibition by sb was recently shown to stimulate forward and hyperactivated motility of human sperm. the mechanism by which p mapk controls ciliary beat is not known. active p mapk could attenuate ciliary beat either by direct or indirect interactions involving phosphorylation of axonemal components or components within a ciliary signal transduction cascade, respectively. studies with c. reinhardtii have demonstrated that phosphorylation and dephosphorylation control flagella motility and have highlighted the complex nature of protein phosphorylation in this organelle. many potential kinase substrates exist that co-ordinate ciliary motility including the dyneins and central pair kinesin klp, but further characterisation of kinase substrates within cilia is needed. only a few other kinases have been reported to regulate cilia beat. pka has been shown to attach to paramecium ciliary axonemes, and strong evidence exists for pka playing a positive role in paramecium swimming and in controlling ciliary beat frequency of mammalian cilia. interestingly, pka has also been implicated in regulating the ciliary motion of s. mansoni miracidia. while cgmp-dependent protein kinase has also been shown to positively regulate ciliary beat frequency, like for p mapk, pkc has been implicated in slowing ciliary beat. clearly, more research is needed into the influence of kinase activities on ciliary movement. for example, erk1/ has recently been found to bind radial spoke protein in mammals and regulate its interaction with pka; however, the extent to which erk actually influences motility via this interaction warrants investigation. activation of p mapk accelerates loss of cilia during larval transformation when miracidia penetrate a suitable intermediate snail host they shed their ciliated plates and develop into the next larval stage, the mother sporocyst. this transformation can also be performed in vitro during which miracidia stop swimming before the plates are released. as p mapk activation attenuates miracidia swim speed whereas inhibition accelerates it we explored the effects of anisomycin and sb on deciliation and thus swimming during miracidium-to-mother sporocyst transformation in vitro. dmso did not affect the deciliation rate when compared to chernin's balanced salt solution controls and none of the treatments affected the survival of the developing larvae. anisomycin accelerated the shedding of ciliary plates considerably; after only h transformation % of parasites had stopped swimming having shed at least some cilia in contrast to only % of parasites in the cbss control group. at this time point % of anisomycin-treated larvae had shed all their ciliated plates compared to none in cbss alone. this effect of anisomycin persisted throughout larval transformation. although the effects of sb were somewhat less marked, at h, h and h significantly more miracidia were observed swimming than were present in the cbss control group. thus, p mapk activation appears to accelerate the initial rate of s. mansoni miracidium transformation by attenuating cilia-mediated swimming behaviour leading to early release of ciliated plates.  here biochemical, innunohistochemical and functional data are presented that are consistent with p mapk playing an important part in the regulation of ciliary beat and thus swimming behaviour of the multicellular eukaryote, s. mansoni. the marked difference in p mapk activation between un-hatched or stationary miracidia and actively swimming miracidia is striking. localization of active p mapk to both the cilium shaft and the tegument of stationary miracidia implies that p mapk might play multiple parts in co-ordinating swim behaviour, including sensory roles as described/hypothesized for motile cilia in other organisms including parasites. given the conservation of both signalling processes and structure/function of motile cilia, we hypothesize that p mapk might regulate ciliary beat frequency in a variety of metazoans. thus our findings could have implications for studies into motility of other important multicellular eukaryotes including parasites of humans, and for research into various human ciliopathies. 
although tremendous progress has been made to comprehend the biochemical basis of these complex evolutionarily-conserved organelles, few protein kinases have been reported to co-ordinate ciliary beat. here we present evidence for p mitogen-activated protein kinase playing a role in the ciliary beat of a multicellular eukaryote, the free-living miracidium stage of the platyhelminth parasite schistosoma mansoni. motile cilia are essential to the survival and reproduction of many eukaryotes; they are responsible for powering swimming of protists and small multicellular organisms and drive fluids across respiratory and reproductive surfaces in mammals. finally, by inhibiting swimming, p mapk activation resulted in early release of ciliated epidermal plates from the miracidium thus accelerating development to the post-miracidium larval stage. western blotting and immunocytochemistry demonstrated that treatment of miracidia with the p mapk activator anisomycin resulted in a rapid, sustained, activation of p mapk, which was primarily localized to the cilia associated with the ciliated epidermal plates, and the tegument.
 motile cilia are essential to the survival and reproduction of many eukaryotes; they are responsible for powering swimming of protists and small multicellular organisms and drive fluids across respiratory and reproductive surfaces in mammals. although tremendous progress has been made to comprehend the biochemical basis of these complex evolutionarily-conserved organelles, few protein kinases have been reported to co-ordinate ciliary beat. here we present evidence for p mitogen-activated protein kinase playing a role in the ciliary beat of a multicellular eukaryote, the free-living miracidium stage of the platyhelminth parasite schistosoma mansoni.  fluorescence confocal microscopy revealed that non-motile miracidia trapped within eggs prior to hatching displayed phosphorylated p mapk associated with their ciliated surface. in contrast, freshly-hatched, rapidly swimming, miracidia lacked phosphorylated p mapk. western blotting and immunocytochemistry demonstrated that treatment of miracidia with the p mapk activator anisomycin resulted in a rapid, sustained, activation of p mapk, which was primarily localized to the cilia associated with the ciliated epidermal plates, and the tegument. freshly-hatched miracidia possessed swim velocities between - mm/s. strikingly, anisomycin-mediated p mapk activation rapidly attenuated swimming, reducing swim velocities by 55% after min and 99% after min. in contrast, sb a p mapk inhibitor, increased swim velocity by up to 15% over this duration. finally, by inhibiting swimming, p mapk activation resulted in early release of ciliated epidermal plates from the miracidium thus accelerating development to the post-miracidium larval stage.  this study supports a role for p mapk in the regulation of ciliary-beat. given the evolutionary conservation of signalling processes and cilia structure, we hypothesize that p mapk may regulate ciliary beat and beat-frequency in a variety of eukaryotes. 