BACKGROUND
a diversity of organisms, ranging from bacteria to humans, shows circadian rhythms in physiology and behavior that are controlled by endogenous oscillators.
in mammals and flies, the core clocks are generated by two negative feedback loops that are interconnected to the same two positive basic helix-loop-helix /pas-containing transcription factors clock  and cycle  .
in d. melanogaster, clk and cyc form a heterodimer that binds to upstream e-box sequences  in period  and timeless , which in turn control their own expression by negatively regulating clk/cyc mediated activation  <cit> .
in the second loop, the products of vrille  and par domain protein  <dig> epsilon , which are also activated by clk/cyc, regulate clk transcription by competing for the same site in its promoter.
whereas vri represses clk production just after lights off, pdp1ε activates it in the middle of the night, separating the phases of clk transcription and repression  <cit> .
these oscillations of gene expression and posttranslational regulation are necessary for the robustness and accuracy of overt physiological and behavioral rhythms.
although the core clock molecules are relatively conserved between mammals and d. melanogaster, there are some interesting differences in, for example, the transcriptional control of clk and cyc expression.
in the suprachiasmatic nuclei  of mammals , clk is constitutively expressed  <cit>  and bmal <dig> is rhythmic, reaching its maximum abundance at dawn  <cit> .
in contrast, cyc is constitutively expressed in d. melanogaster heads  <cit> , while clk shows rhythmic expression on the mrna level, peaking during the night-day transition   <cit> .
although the drosophila clk protein has also been reported to cycle with the same phase of its mrna  <cit> , recent data indicates that was a result of a methodological artifact  <cit> .
its ability to bind e-boxes and activate transcription in a cyclic manner in fact resides in its phosphorylation pattern, with only the late day/early night hypophosphorylated forms being capable of promoting per and tim expression  <cit> .
the molecular study of circadian rhythms in insect vectors is still in its infancy.
in sandflies the circadian expression profiles of per, tim and clk has been studied in lutzomyia longipalpis, the main vector of visceral leishmaniasis in the americas  <cit> .
while per and tim cycle as in other insects, peaking around zt  <dig>  <cit> , clk expression peaks around zt 9– <dig>  about half a day later than in d. melanogaster  <cit> .
this difference in clk expression is correlated with differences in locomotor activity.
drosophila shows a bimodal/diurnal pattern, whereas lutzomyia is predominately unimodal/nocturnal  <cit> .
in addition, blood feeding causes a reduction in sandfly locomotor activity that is accompanied by a reduction in per and tim, but not clk levels  <cit> .
thus, as the clk profile of l. longipalpis is different from that of d. melanogaster, we wondered if the same would occur for its partner cyc.
we therefore cloned the l. longipalpis cyc gene and report here its genomic structure and the putative amino acid sequence.
the presence of an alternative transcript is also identified.
in addition we have analyzed the daily expression of cyc in males and females, as well as its expression after a blood meal.
RESULTS
cloning and sequence analysis
the sequence of the lutzomyia longipalpis cycle gene  was obtained using a pcr gene walking approach.
an initial fragment was obtained using degenerate primers.
based on this first sequence new specific primers were designed and used in new reactions to obtain further sequences.
the 5' and 3' ends were obtained using race techniques .
introns were mapped by comparing pcr fragments generated using cdna and genomic dna as templates.
fig  <dig> shows a schematic representation of the l. longipalpis cyc gene with its seven introns.
the figure also shows the regions coding for the different domains of the protein and the position of an alternative splice form .
l. longipalpis cycle codes for a putative protein of  <dig> amino acids homologous to cyc from other species .
alignment of the sandfly cyc sequence with insect orthologues  and one mammal  reveals high conservation in some particular regions such as the bhlh dna binding domain and the protein dimerization pas a and pas b regions, especially when compared to a. gambiae and d. melanogaster .
in addition, we were able to find in sandfly cyc the bmal <dig> c-terminal region , which was characterized as an activation of the clk/bmal <dig> heterodimer in a mammalian cell culture  <cit>  .
this region in l. longipalpis cyc is  <dig> % identical to the mosquito predicted cyc sequence,  <dig> % identical to the moth a. pernyi and  <dig> % identical to the mouse bmal <dig> sequences .
interestingly, this motif is not present at all in drosophila .
fig  <dig> shows a neighbor-joining tree using the alignment of the protein sequences shown in fig  <dig>  as expected l. longipalpis cyc clustered with the a. gambiae sequence.
the approximate positions of the seven introns of the l. longipalpis cycle gene are also marked in fig  <dig> by inverted triangles.
inspection of cdna and genomic sequences available for anopheles gambiae cyc revealed that only three out of the seven intron positions of l. longipalpis cyc  are conserved between the two species .
comparison of different cdna sequences also revealed the existence of a rare alternative splice transcript missing only one arginine codon .
nevertheless this single difference potentially alters the ability of the putative protein to be phosphorylated .
this minor transcript corresponds to about 20% of all sequenced cdna fragments .
we also searched in sandfly cyc sequence possible conserved residues for pos-translational modification .
we were able to find a lysine at the position lys- <dig> in a conserved sumo consensus site  in the pas link region, at an approximate position where its mammalian homologue is sumoylated in vivo   <cit>  .
in addition we were able to find ser- <dig> and a thr- <dig> at conserved positions, corresponding to the mammalian ser- <dig> and thr- <dig>  which are phosphorylated in vitro  <cit> .
this region was further examined for potential kinase substrate sites using scansite  <dig>    <cit>  and the ser- <dig> was identified as potential casein kinase- <dig> phosphorylation site  .
interestingly, the rare alternative transcript identified as missing a single arg did not reach significance in the phosphorylation prediction at high stringency, suggesting another level of sandfly cyc regulation.
temporal cyc expression analysis
as per mrna levels are differentially expressed between the head and body in females of d. melanogaster, female sandfly heads and bodies were analyzed separately.
sandfly males were not dissected since no differences are observed in per expression between heads and bodies in drosophila  <cit> .
analysis of l. longipalpis cyc expression in males and female heads under ld12: <dig> relative to the rp <dig> constitutive control indicates clear cycling in mrna abundance.
as for sandfly per, tim and clk  <cit>  no significant differences between males and female heads were observed, and therefore these results were pooled.
anova indicates that cyc mrna levels are significantly different among zt groups  with a peak around zt 5– <dig>  with levels over two-fold higher compared to the levels at zt  <dig>  .
this is similar to the observed bmal <dig> cycling in mammals  <cit>  but different compared to most insects analyzed to date, where its expression is constitutive  <cit> .
on the other hand, in female bodies, cyc expression was constitutive  , in contrast to bmal <dig> expression in all mammalian tissues analyzed so far  <cit> .
cyc expression analysis in blood-fed females
in order to know if blood feeding has an effect on cyc expression as it does for per and tim  <cit> , we assayed its mrna levels  <dig> hs after a full blood meal.
but, as for clk  <cit> , cyc levels were not significantly altered in heads and bodies of ingurgitated females compared to unfed controls .
discussion
in this study we characterized the sequence, genomic structure and expression of the cycle gene in the hematophagous sandfly l. longipalpis.
analysis of predicted protein sequence revealed its homology with cyc from others species .
interestingly, the bmal <dig> c-terminal region , which was characterized as responsible for the activation of the clk/bmal <dig> heterodimer in a mammalian cell culture  <cit> , was also found in sandfly cyc.
the conservation of this region in all animals analyzed so far  suggests that sandfly cyc may also possess a c-terminal transactivation domain  <cit> .
chang et al  <cit>  studying moth clock genes have suggested that the bctr is very ancient, being lost in drosophila cyc probably because it became redundant after the fruitfly clk had acquired a new transactivation domain, a large poly-q region.
this latter domain is not found in the moth clk orthologue and we are currently cloning the sandfly clk to determine if the same is true for this vector species.
an important feature of mammalian cyc regulation is the phosphorylation and sumoylation of its serine/threonine and lysine residues respectively  <cit> .
aligning cyc homologues from different species we were able to find a lysine in the pas link region of sandfly cyc at an approximately similar position where its homologue in mammals is sumoylated .
in addition, prediction phosphorylation site analysis identified ser- <dig> as a potential target for posttranslational modification, but only in the more abundant form.
in the alternative transcript identified, the missing arg alters the ability of the ser to be phosphorylated.
this difference is noteworthy since in mammals only the hypophosphorylated form is able to bind to e-boxes in vitro, showing that phosphorylation of bmal <dig> might play an important role in pacemaker regulation  <cit> .
taken together, these results suggest that sandfly cyc might be regulated at different levels , which may be important for its role in the sandfly pacemaker.
our results on daily gene expression in males and female heads, unexpectedly, resemble data from mammals where cyc expression is also rhythmic   <cit> .
unlike most insects analyzed so far  sandfly cyc cycled robustly, beginning to rise at the end of the night  and peaking in the middle of the day zt 5– <dig> .
in drosophila posttranslational mechanisms are necessary to provide optimal levels and subcellular localization of clock proteins.
earlier data have indicated that per and tim start to accumulate when clk levels are decreasing  <cit> , and this cannot be satisfactorily explained by a simple feedback loop model  <cit> .
this contradiction was recently clarified by two papers that show that clk levels in fact do not cycle  <cit> .
nevertheless, clk transcriptional activity is rhythmic, via its phosphorylation levels.
while hyperphosphorylated clk predominates during times of transcriptional repression , hypophosphorylated clk is more abundant during times of transcriptional activation   <cit> .
the authors of these studies suggest that hypophosphorylated clk forms complexes with cyc at midday, bind to e-boxes and initiate per and tim transcription.
once the tim/per/dbt complex enters the nucleus it represses transcription by inhibiting clk/cyc e-box binding and promoting clk hyperphosphorylation and degradation  <cit> .
on the other hand our previous report on per, tim and clk expression in sandflies satisfied a simple feedback loop model, since per and tim levels rise at the time when clk levels reaches its peak  <cit> .
given that in head oscillators cyc expression is earlier than clk and that we identified at least one strong putative motif for phosphorylation, we propose that sandfly cyc might be subject to posttranslational modification, which would provide the necessary time delay for its accumulation at the appropriate time of day .
in contrast to heads, cyc expression in female bodies was shown to be constitutive .
in drosophila per was shown to be constitutively expressed in ovaries  <cit>  causing a strong damping in per cycling in female bodies.
in fact, sandfly per is also constitutive in female bodies  <cit> .
the differential regulation of cyc through the sandfly body suggests that, as in drosophila and mammals  <cit> , clock genes in l. longipalpis may also play different roles in different tissues, reflecting particular interactions with different molecules, what would finally lead to the coordination of other aspects of sandfly physiology.
interestingly the mammalian orthologue bmal <dig> was shown to interact with non-circadian transcription factors, which in turn could respond to different kinds of stimuli  <cit> .
finally, data on blood-fed females shows that, although per and tim expression are downregulated, clk and cyc are not .
since the latter two are activators of the formers, we believe that blood-feeding might regulate negatively clk and cyc function at the posttranscriptional level, leading to diminished per and tim activation.
this could be mediated by changes in nadh/nad+ levels, which can be altered by blood-feeding in other insect species  <cit> .
furthermore, changes in redox state have been observed to alter mammalian clk activity in vitro  <cit> .
this latter observation is consistent with the observations that feeding and fasting, which would be expected to change the redox profile, can entrain mammalian peripheral clocks independently of the ld cycles  <cit> .
however, restricted-feeding regimes in drosophila do not appear to influence circadian behavior or molecular rhythms of per and tim  <cit> .
CONCLUSIONS
the present results, together with our previous data, show that the molecular clock of l. longipalpis shows interesting differences compared to drosophila, suggesting that blood-sucking insect vector species might present very interesting comparative models to study circadian rhythms and its molecular control.
in addition, since the circadian clock drives activity and feeding behavior in insect vectors, understanding the molecular machinery of the clock may add important information in the dynamics of vector-borne disease transmission.

 a diversity of organisms, ranging from bacteria to humans, shows circadian rhythms in physiology and behavior that are controlled by endogenous oscillators. in mammals and flies, the core clocks are generated by two negative feedback loops that are interconnected to the same two positive basic helix-loop-helix /pas-containing transcription factors clock and cycle. in d. melanogaster, clk and cyc form a heterodimer that binds to upstream e-box sequences in period and timeless, which in turn control their own expression by negatively regulating clk/cyc mediated activation. in the second loop, the products of vrille and par domain protein epsilon, which are also activated by clk/cyc, regulate clk transcription by competing for the same site in its promoter. whereas vri represses clk production just after lights off, pdp1ε activates it in the middle of the night, separating the phases of clk transcription and repression. these oscillations of gene expression and posttranslational regulation are necessary for the robustness and accuracy of overt physiological and behavioral rhythms. although the core clock molecules are relatively conserved between mammals and d. melanogaster, there are some interesting differences in, for example, the transcriptional control of clk and cyc expression. in the suprachiasmatic nuclei of mammals, clk is constitutively expressed and bmal is rhythmic, reaching its maximum abundance at dawn. in contrast, cyc is constitutively expressed in d. melanogaster heads, while clk shows rhythmic expression on the mrna level, peaking during the night-day transition. although the drosophila clk protein has also been reported to cycle with the same phase of its mrna, recent data indicates that was a result of a methodological artifact. its ability to bind e-boxes and activate transcription in a cyclic manner in fact resides in its phosphorylation pattern, with only the late day/early night hypophosphorylated forms being capable of promoting per and tim expression. the molecular study of circadian rhythms in insect vectors is still in its infancy. in sandflies the circadian expression profiles of per, tim and clk has been studied in lutzomyia longipalpis, the main vector of visceral leishmaniasis in the americas. while per and tim cycle as in other insects, peaking around zt, clk expression peaks around zt 9– about half a day later than in d. melanogaster. this difference in clk expression is correlated with differences in locomotor activity. drosophila shows a bimodal/diurnal pattern, whereas lutzomyia is predominately unimodal/nocturnal. in addition, blood feeding causes a reduction in sandfly locomotor activity that is accompanied by a reduction in per and tim, but not clk levels. thus, as the clk profile of l. longipalpis is different from that of d. melanogaster, we wondered if the same would occur for its partner cyc. we therefore cloned the l. longipalpis cyc gene and report here its genomic structure and the putative amino acid sequence. the presence of an alternative transcript is also identified. in addition we have analyzed the daily expression of cyc in males and females, as well as its expression after a blood meal.  cloning and sequence analysis the sequence of the lutzomyia longipalpis cycle gene was obtained using a pcr gene walking approach. an initial fragment was obtained using degenerate primers. based on this first sequence new specific primers were designed and used in new reactions to obtain further sequences. the 5' and 3' ends were obtained using race techniques. introns were mapped by comparing pcr fragments generated using cdna and genomic dna as templates. fig shows a schematic representation of the l. longipalpis cyc gene with its seven introns. the figure also shows the regions coding for the different domains of the protein and the position of an alternative splice form. l. longipalpis cycle codes for a putative protein of amino acids homologous to cyc from other species. alignment of the sandfly cyc sequence with insect orthologues and one mammal reveals high conservation in some particular regions such as the bhlh dna binding domain and the protein dimerization pas a and pas b regions, especially when compared to a. gambiae and d. melanogaster. in addition, we were able to find in sandfly cyc the bmal c-terminal region, which was characterized as an activation of the clk/bmal heterodimer in a mammalian cell culture. this region in l. longipalpis cyc is % identical to the mosquito predicted cyc sequence, % identical to the moth a. pernyi and % identical to the mouse bmal sequences. interestingly, this motif is not present at all in drosophila. fig shows a neighbor-joining tree using the alignment of the protein sequences shown in fig as expected l. longipalpis cyc clustered with the a. gambiae sequence. the approximate positions of the seven introns of the l. longipalpis cycle gene are also marked in fig by inverted triangles. inspection of cdna and genomic sequences available for anopheles gambiae cyc revealed that only three out of the seven intron positions of l. longipalpis cyc are conserved between the two species. comparison of different cdna sequences also revealed the existence of a rare alternative splice transcript missing only one arginine codon. nevertheless this single difference potentially alters the ability of the putative protein to be phosphorylated. this minor transcript corresponds to about 20% of all sequenced cdna fragments. we also searched in sandfly cyc sequence possible conserved residues for pos-translational modification. we were able to find a lysine at the position lys- in a conserved sumo consensus site in the pas link region, at an approximate position where its mammalian homologue is sumoylated in vivo. in addition we were able to find ser- and a thr- at conserved positions, corresponding to the mammalian ser- and thr- which are phosphorylated in vitro. this region was further examined for potential kinase substrate sites using scansite and the ser- was identified as potential casein kinase- phosphorylation site. interestingly, the rare alternative transcript identified as missing a single arg did not reach significance in the phosphorylation prediction at high stringency, suggesting another level of sandfly cyc regulation. temporal cyc expression analysis as per mrna levels are differentially expressed between the head and body in females of d. melanogaster, female sandfly heads and bodies were analyzed separately. sandfly males were not dissected since no differences are observed in per expression between heads and bodies in drosophila. analysis of l. longipalpis cyc expression in males and female heads under ld12: relative to the rp constitutive control indicates clear cycling in mrna abundance. as for sandfly per, tim and clk no significant differences between males and female heads were observed, and therefore these results were pooled. anova indicates that cyc mrna levels are significantly different among zt groups with a peak around zt 5– with levels over two-fold higher compared to the levels at zt. this is similar to the observed bmal cycling in mammals but different compared to most insects analyzed to date, where its expression is constitutive. on the other hand, in female bodies, cyc expression was constitutive, in contrast to bmal expression in all mammalian tissues analyzed so far. cyc expression analysis in blood-fed females in order to know if blood feeding has an effect on cyc expression as it does for per and tim, we assayed its mrna levels hs after a full blood meal. but, as for clk, cyc levels were not significantly altered in heads and bodies of ingurgitated females compared to unfed controls. discussion in this study we characterized the sequence, genomic structure and expression of the cycle gene in the hematophagous sandfly l. longipalpis. analysis of predicted protein sequence revealed its homology with cyc from others species. interestingly, the bmal c-terminal region, which was characterized as responsible for the activation of the clk/bmal heterodimer in a mammalian cell culture, was also found in sandfly cyc. the conservation of this region in all animals analyzed so far suggests that sandfly cyc may also possess a c-terminal transactivation domain. chang et al studying moth clock genes have suggested that the bctr is very ancient, being lost in drosophila cyc probably because it became redundant after the fruitfly clk had acquired a new transactivation domain, a large poly-q region. this latter domain is not found in the moth clk orthologue and we are currently cloning the sandfly clk to determine if the same is true for this vector species. an important feature of mammalian cyc regulation is the phosphorylation and sumoylation of its serine/threonine and lysine residues respectively. aligning cyc homologues from different species we were able to find a lysine in the pas link region of sandfly cyc at an approximately similar position where its homologue in mammals is sumoylated. in addition, prediction phosphorylation site analysis identified ser- as a potential target for posttranslational modification, but only in the more abundant form. in the alternative transcript identified, the missing arg alters the ability of the ser to be phosphorylated. this difference is noteworthy since in mammals only the hypophosphorylated form is able to bind to e-boxes in vitro, showing that phosphorylation of bmal might play an important role in pacemaker regulation. taken together, these results suggest that sandfly cyc might be regulated at different levels, which may be important for its role in the sandfly pacemaker. our results on daily gene expression in males and female heads, unexpectedly, resemble data from mammals where cyc expression is also rhythmic. unlike most insects analyzed so far sandfly cyc cycled robustly, beginning to rise at the end of the night and peaking in the middle of the day zt 5–. in drosophila posttranslational mechanisms are necessary to provide optimal levels and subcellular localization of clock proteins. earlier data have indicated that per and tim start to accumulate when clk levels are decreasing, and this cannot be satisfactorily explained by a simple feedback loop model. this contradiction was recently clarified by two papers that show that clk levels in fact do not cycle. nevertheless, clk transcriptional activity is rhythmic, via its phosphorylation levels. while hyperphosphorylated clk predominates during times of transcriptional repression, hypophosphorylated clk is more abundant during times of transcriptional activation. the authors of these studies suggest that hypophosphorylated clk forms complexes with cyc at midday, bind to e-boxes and initiate per and tim transcription. once the tim/per/dbt complex enters the nucleus it represses transcription by inhibiting clk/cyc e-box binding and promoting clk hyperphosphorylation and degradation. on the other hand our previous report on per, tim and clk expression in sandflies satisfied a simple feedback loop model, since per and tim levels rise at the time when clk levels reaches its peak. given that in head oscillators cyc expression is earlier than clk and that we identified at least one strong putative motif for phosphorylation, we propose that sandfly cyc might be subject to posttranslational modification, which would provide the necessary time delay for its accumulation at the appropriate time of day. in contrast to heads, cyc expression in female bodies was shown to be constitutive. in drosophila per was shown to be constitutively expressed in ovaries causing a strong damping in per cycling in female bodies. in fact, sandfly per is also constitutive in female bodies. the differential regulation of cyc through the sandfly body suggests that, as in drosophila and mammals, clock genes in l. longipalpis may also play different roles in different tissues, reflecting particular interactions with different molecules, what would finally lead to the coordination of other aspects of sandfly physiology. interestingly the mammalian orthologue bmal was shown to interact with non-circadian transcription factors, which in turn could respond to different kinds of stimuli. finally, data on blood-fed females shows that, although per and tim expression are downregulated, clk and cyc are not. since the latter two are activators of the formers, we believe that blood-feeding might regulate negatively clk and cyc function at the posttranscriptional level, leading to diminished per and tim activation. this could be mediated by changes in nadh/nad+ levels, which can be altered by blood-feeding in other insect species. furthermore, changes in redox state have been observed to alter mammalian clk activity in vitro. this latter observation is consistent with the observations that feeding and fasting, which would be expected to change the redox profile, can entrain mammalian peripheral clocks independently of the ld cycles. however, restricted-feeding regimes in drosophila do not appear to influence circadian behavior or molecular rhythms of per and tim.  the present results, together with our previous data, show that the molecular clock of l. longipalpis shows interesting differences compared to drosophila, suggesting that blood-sucking insect vector species might present very interesting comparative models to study circadian rhythms and its molecular control. in addition, since the circadian clock drives activity and feeding behavior in insect vectors, understanding the molecular machinery of the clock may add important information in the dynamics of vector-borne disease transmission. 
here we describe the sequence, genomic organization and circadian expression of cycle in the crepuscular/nocturnal hematophagous sandfly lutzomyia longipalpis, the main vector of visceral leishmaniasis in the americas. unlike drosophila, the molecular clock of insect vectors is poorly understood, despite the importance of circadian behavior in the dynamic of disease transmission. sequence and expression analysis of cycle in l. longipalpis show interesting differences compared to drosophila suggesting that hematophagous vector species might present interesting new models to study the molecular control of insect circadian clocks. deduced amino acid sequence revealed that sandfly cycle has a c-terminal transactivation domain highly conserved among eukaryotes but absent in d. melanogaster. interestingly, while cycle expression in drosophila and other diptera is constitutive, in sandflies it is rhythmic in males and female heads but constitutive in the female body.
 a large number of organisms have internal circadian clocks that enable them to adapt to the cyclic changes of the external environment. in the model organism drosophila melanogaster, feedback loops of transcription and translation are believed to be crucial for the maintenance of the central pacemaker. in this mechanism the cycle gene, which is constitutively expressed, plays a critical role activating the expression of genes that will later inhibit their own activity, thereby closing the loop. unlike drosophila, the molecular clock of insect vectors is poorly understood, despite the importance of circadian behavior in the dynamic of disease transmission.  here we describe the sequence, genomic organization and circadian expression of cycle in the crepuscular/nocturnal hematophagous sandfly lutzomyia longipalpis, the main vector of visceral leishmaniasis in the americas. deduced amino acid sequence revealed that sandfly cycle has a c-terminal transactivation domain highly conserved among eukaryotes but absent in d. melanogaster. moreover, an alternative form of the transcript was also identified. interestingly, while cycle expression in drosophila and other diptera is constitutive, in sandflies it is rhythmic in males and female heads but constitutive in the female body. blood-feeding, which causes down-regulation of period and timeless in this species, does not affect cycle expression.  sequence and expression analysis of cycle in l. longipalpis show interesting differences compared to drosophila suggesting that hematophagous vector species might present interesting new models to study the molecular control of insect circadian clocks. 