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 (bHLH)/PAS-containing transcription factors CLOCK (CLK) and CYCLE (CYC) (also called BMAL1)(reviewed in [1,2]).

InD. melanogaster, CLK and CYC form a heterodimer that binds to upstream E-box sequences (CACGTG) inperiod (per) andtimeless (tim), which in turn control their own expression by negatively regulating CLK/CYC mediated activation [1,2]. In the second loop, the products ofvrille (vri) andPAR domain protein 1 epsilon (Pdp1 ε), which are also activated by CLK/CYC, regulateClk transcription by competing for the same site in its promoter. Whereas VRI repressesClk production just after lights off, PDP1ε activates it in the middle of the night, separating the phases ofClk transcription and repression [3,4]. 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 andD. melanogaster, there are some interesting differences in, for example, the transcriptional control ofClk andcyc expression. In the suprachiasmatic nuclei (SCN) of mammals (where the central pacemaker is located),Clk is constitutively expressed [5] andBmal1 is rhythmic, reaching its maximum abundance at dawn [6,7]. In contrast,cyc is constitutively expressed inD. melanogaster heads [8,9], whileClk shows rhythmic expression on the mRNA level, peaking during the night-day transition (ZT 22-2) [10,11]. Although theDrosophila CLK protein has also been reported to cycle with the same phase of its mRNA [10,11], recent data indicates that was a result of a methodological artifact [12,13]. 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 promotingper andtim expression [12,13].

The molecular study of circadian rhythms in insect vectors is still in its infancy. In sandflies the circadian expression profiles ofper,tim andClk has been studied inLutzomyia longipalpis, the main vector of visceral leishmaniasis in the Americas [14]. Whileper andtim cycle as in other insects, peaking around ZT 13 [15–17],Clk expression peaks around ZT 9–13, about half a day later than inD. melanogaster [10,11,14]. This difference inClk expression is correlated with differences in locomotor activity.Drosophila shows a bimodal/diurnal pattern, whereasLutzomyia is predominately unimodal/nocturnal [14]. In addition, blood feeding causes a reduction in sandfly locomotor activity that is accompanied by a reduction inper andtim, but notClk levels [14]. Thus, as theClk profile ofL. longipalpis is different from that ofD. melanogaster, we wondered if the same would occur for its partnercyc.

We therefore cloned theL. 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 ofcyc in males and females, as well as its expression after a blood meal.

Cloning and sequence analysis

The sequence of theLutzomyia longipalpis cycle gene (GenBank accession number DQ841151) 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 (see Methods for details). Introns were mapped by comparing PCR fragments generated using cDNA and genomic DNA as templates. Fig1 shows a schematic representation of theL. 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 (see below).

Genomic structureL. longipalpis cyclegene. Schematic representation of the genomic structureL. longipalpis cycle gene. The introns are numbered and were drawn to scale. The first and the second introns are depicted by a broken line to indicate that their complete sequence has not been determined. The coding region of theL. longipalpis cycle gene consists of 8 exons and 7 introns and the conserved motifs are shown in blue (bHLH), green (PAS A and B respectively) and yellow (BCTR). In red is marked the approximate position of the alternatively spliced Arg (see text).

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L. longipalpis cycle codes for a putative protein of 622 amino acids homologous to CYC from other species (Fig2). Alignment of the sandfly CYC sequence with insect orthologues (Anopheles gambiae,D. melanogaster,Bombyx mori andAntheraea pernyi) and one mammal (Mus musculus) 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 toA. gambiae andD. melanogaster (see Table1). In addition, we were able to find in sandfly CYC the BMAL1 C-terminal region ("BCTR"), which was characterized as an activation of the CLK/BMAL1 heterodimer in a mammalian cell culture [18] (Fig2). This region inL. longipalpis CYC is 96.87% identical to the mosquito predicted CYC sequence, 87.5% identical to the mothA. pernyi and 93.75% identical to the mouse BMAL1 sequences (Table1). Interestingly, this motif is not present at all inDrosophila (Fig2; see Discussion). Fig3 shows a Neighbor-joining tree using the alignment of the protein sequences shown in Fig2. As expectedL. longipalpis CYC clustered with theA. gambiae sequence.

Alignment of CYCLE protein sequences. Protein alignment of theL. longipalpis CYCLE sequence with orthologues fromAnopheles gambiae (Ensembl: ENSANGP00000027387),Drosophila melanogaster (GenBank™: AAC39124),Bombyx mori (GenBank™: BAB91178),Antheraea pernyi (GenBank™: AAR14937) andMus musculus (GenBank™: AAH25973). The marked motifs use the same colors as in Fig 1.

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Neighbor-joining tree of CYCLE protein sequences. Neighbor-joining tree using the alignment of the protein sequences shown in Fig 2, complete deletion and Poisson corrected distances.Mus musculus was used as the outgroup.

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The approximate positions of the seven introns of theL. longipalpis cycle gene are also marked in fig2 by inverted triangles. Inspection of cDNA and genomic sequences available forAnopheles gambiae cyc revealed that only three out of the seven intron positions ofL. longipalpis cyc (2, 3 and 7) are conserved between the two species (data not shown). Comparison of different cDNA sequences also revealed the existence of a rare alternative splice transcript missing only one Arginine codon (Fig1 and2). Nevertheless this single difference potentially alters the ability of the putative protein to be phosphorylated (see below). This minor transcript corresponds to about 20% of all sequenced cDNA fragments (7/35).

We also searched in sandfly CYC sequence possible conserved residues for pos-translational modification (Fig4a,b). We were able to find a lysine at the position Lys-225 in a conserved sumo consensus site (ΨKXE/D, where Ψ is a hydrophobic residue and X may be any amino acid) in the PAS link region, at an approximate position where its mammalian homologue is sumoylatedin vivo (Lys-259) [19] (Fig4a). In addition we were able to find Ser-502 and a Thr-510 at conserved positions, corresponding to the mammalian Ser-527 and Thr-534, which are phosphorylatedin vitro [20]. This region was further examined for potential kinase substrate sites using Scansite 2.0 (stringency levels = high) [21] and the Ser-502 was identified as potential casein kinase-1 phosphorylation site (Score: 0.3474; Percentile: 0.081%) (Fig4b). 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.

Potential Sumo modification and Phosphorylation sites in theL. longipalpisCYCLE sequence. Potential Sumo modification and Phosphorylation sites in theL. longipalpis CYCLE sequence. (A) Alignment of theM. musculus,A. gambiae,D. melanogaster andL. longipalpis PAS linker region. The three insect species listed contains a potential SUMO consensus motif and the correspondent Lys that is sumoylated in mammals. (B) A Ser-rich region (Ser underlined) is shown forM. musculus,A. gambiae andL. longipalpis CYC. Ser and Thr residues are phosphorylated in mammals and their potential cognates inL. longipalpis andA. gambiae CYC are highlighted in yellow. In red is marked the position of the alternatively spliced Arg (see text).

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Temporalcyc expression analysis

Asper mRNA levels are differentially expressed between the head and body in females ofD. melanogaster, female sandfly heads and bodies were analyzed separately. Sandfly males were not dissected since no differences are observed inper expression between heads and bodies inDrosophila [22].

Analysis ofL. longipalpis cyc expression in males and female heads under LD12:12 relative to therp49 constitutive control indicates clear cycling in mRNA abundance. As for sandflyper,tim andClk [14] no significant differences between males and female heads were observed, and therefore these results were pooled. ANOVA indicates thatcyc mRNA levels are significantly different among ZT groups (F_5,72 = 4.481; P < 0.001) with a peak around ZT 5–9, with levels over two-fold higher compared to the levels at ZT 17 (P < 0.001, LSD – Least significant difference analysis) (Fig5a). This is similar to the observedBmal1 cycling in mammals [6,7] but different compared to most insects analyzed to date, where its expression is constitutive [8,9,16,23]. On the other hand, in female bodies,cyc expression was constitutive (F_5,34 = 0.086; P = 0.994) (Fig5b), in contrast toBmal1 expression in all mammalian tissues analyzed so far [24,25].

Daily expression ofL. longipalpis cycle. Daily expression ofL. longipalpis cycle. A. Pooled data for males and female heads. B. Female bodies. Bars represent the mean relative abundance +/- the range based on the SEM (Standard Error of the Mean).

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cyc expression analysis in blood-fed females

In order to know if blood feeding has an effect oncyc expression as it does forper andtim [14], we assayed its mRNA levels 27 hs after a full blood meal. But, as forClk [14],cyc levels were not significantly altered in heads and bodies of ingurgitated females compared to unfed controls (Fig6).

cycleexpression in unfed and blood-fedL. longipalpisfemales. Relative expression ofcycle in unfed and blood-fedL. longipalpis female heads (top) and bodies (bottom). Columns (+/- the range based on the SEM) represent the normalized relative abundance.

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In this study we characterized the sequence, genomic structure and expression of thecycle gene in the hematophagous sandflyL. longipalpis. Analysis of predicted protein sequence revealed its homology withcyc from others species (Fig2). Interestingly, the BMAL1 C-terminal region ("BCTR"), which was characterized as responsible for the activation of the CLK/BMAL1 heterodimer in a mammalian cell culture [18], was also found in sandfly CYC. The conservation of this region in all animals analyzed so far (exceptDrosophila) suggests that sandfly CYC may also possess a C-terminal transactivation domain [8,23,26,27]. Chang et al [26] studying moth clock genes have suggested that the BCTR is very ancient, being lost inDrosophila 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 sandflyClk 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 [19,20,28]. 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 (Fig4a). In addition, prediction phosphorylation site analysis identified Ser-502 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-boxesin vitro, showing that phosphorylation of BMAL1 might play an important role in pacemaker regulation [20,28]. Taken together, these results suggest that sandfly CYC might be regulated at different levels (transcriptional and posttranslational), 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 wherecyc expression is also rhythmic (Fig5a) [6,7,24]. Unlike most insects analyzed so far (where no oscillation ofcyc mRNA was detected [9,16,23], but see Rubin et al [27]) sandflycyc cycled robustly, beginning to rise at the end of the night (ZT 21) and peaking in the middle of the day ZT 5–9 (Fig5a).

InDrosophila posttranslational mechanisms are necessary to provide optimal levels and subcellular localization of clock proteins. Earlier data have indicated thatper andtim start to accumulate when CLK levels are decreasing [10,11], and this cannot be satisfactorily explained by a simple feedback loop model [1,2]. This contradiction was recently clarified by two papers that show that CLK levels in fact do not cycle [12,13]. Nevertheless, CLK transcriptional activity is rhythmic, via its phosphorylation levels. While hyperphosphorylated CLK predominates during times of transcriptional repression (late night/early morning), hypophosphorylated CLK is more abundant during times of transcriptional activation (late day/early night) [12,13]. The authors of these studies suggest that hypophosphorylated CLK forms complexes with CYC at midday, bind to E-boxes and initiateper andtim 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 [12,13]. On the other hand our previous report onper,tim andClk expression in sandflies satisfied a simple feedback loop model, sinceper andtim levels rise at the time whenClk levels reaches its peak [14]. Given that in head oscillatorscyc expression is earlier thanClk 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 (ZT 13, when it can dimerize with the product ofClk and driveper andtim transcription [9,14]).

In contrast to heads,cyc expression in female bodies was shown to be constitutive (Fig5b). InDrosophila per was shown to be constitutively expressed in ovaries [22] causing a strong damping inper cycling in female bodies. In fact, sandflyper is also constitutive in female bodies [14]. The differential regulation ofcyc through the sandfly body suggests that, as inDrosophila and mammals [29], clock genes inL. 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 BMAL1 was shown to interact with non-circadian transcription factors, which in turn could respond to different kinds of stimuli [30].

Finally, data on blood-fed females shows that, althoughper andtim expression are downregulated,Clk andcyc are not [[14] and this report]. 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 diminishedper andtim activation. This could be mediated by changes in NAD(P)H/NAD(P)+ levels, which can be altered by blood-feeding in other insect species [31,32]. Furthermore, changes in redox state have been observed to alter mammalian CLK activityin vitro [33]. 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 [34,35]. However, restricted-feeding regimes inDrosophila do not appear to influence circadian behavior or molecular rhythms ofper andtim [36].

The present results, together with our previous data, show that the molecular clock ofL. longipalpis shows interesting differences compared toDrosophila, 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.

Insects

L. longipalpis sandflies from a Lapinha (Minas Gerais State, Brazil) laboratory colony were reared as previously described [14,37]. Briefly, for the temporal gene expression experiments three independent replicate samples with circa 40 sandflies were collected on the fourth day of entrainment at ZTs 1, 5, 9, 13, 17 and 21. Only females were dissected due to their differential pattern of expression between heads and body tissues [14]. For blood-feeding experiments two to three-day-old females were blood-fed on an anaesthetized hamster during 10 min at the light-dark transition. Afterwards blood-fed and unfed controls (from the same cage) were separated and kept in different cages in an incubator at 25°C and LD12:12. Since blood-fed and unfed controls had to be visually separated after the feeding period, they were subjected to a phase-delay of 2 h, that is, placed in a different incubator with lights turning on and off 2 h later than the previous one where they were entrained. They were collected and frozen at ZT 13 in the following day (27 h after the blood meal – 2 h needed to separate blood-fed and unfed controls plus 25 h to reach the ZT 13 in the next day). This procedure was shown not to affect sandfly behavior norper,tim andClk expression [14].

Cloning of sandflycyc

Genomic sandfly DNA from circa 20 individuals was extracted with the GenomicPrep™ (Amersham Biosciences) kit according to manufacturer instructions. A fragment homologous to theDrosophila cyc was first amplified fromL. longipalpis genomic DNA using the degenerate primer PCR technique. The primer sequences were as follows: 5'CYCdeg1, 5'A(A, G)(A, C)GN(A, C)GN(A, C)GNGA(T, C)AA(A, G)ATGAA 3' & 3'CYCdeg1, 5'AC(C, T)TTNCC(A, G, T)AT(A, G)TC(C, T)TTNGG(A, G)TG 3'. Sequential reactions were carried out to reach the 3' and 5' end of the gene as follows. For the missing 5' of the gene we used the "5' Race System for Rapid Amplification of cDNA Ends" kit (Gibco BRL). Primer used in the 1^st strand synthesis 3'llCYCexp1: 5'TTATGGAAGTGGCCATGGGAGTCC 3'. Then the first PCR reaction was done with the primers 5'RACE AAP: 5'GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG 3' & 3'llCYC8: 5'CTCCTTGACCTTAGCCACATC 3'. Reamplification of this material was done with the nested AUAP 5'GGCCACGCGTCGACTAGTAC 3' & 3' llCYC7 5'TGGGAGTAATTGAGGACCTGC 3' primers according to manufacturer instructions. For the 3' region a preliminary reaction with specific and degenerate primers was done before the 3'RACE: initial reaction with primers 5'llCYC2 5'GGTCCTCAATTACTCCCAAG 3' & 3'CYCdeg2 5'TTCATNC(G, T)(A, G)CA(A, G)AA(A, G)AA 3' and later with the primers 5'llCYC3 5'CAATGCTTCCGGTGAAGACG 3' & 3' CYCdeg3 5'(G, C)(A, T)NGTNCCNA(A, G)(A, G, T)AT(C, T)TC(C, T)TG 3'. The 3' extreme end of the gene was obtained with the following primers: 5'llCYC7 5'CAGTTCATCTCTCGTCATGCC 3' & oligo dT and later a nested reaction: 5'llCYC6 5'CGTTGATTCTGGGCTTCCTAC 3' & oligo dT. Gene fragments were cloned in a pMOS vector (Amersham Biosciences) and sequenced at the Department of Biochemistry and Molecular Biology, Instituto Oswaldo Cruz – FIOCRUZ on an ABI 377XL DNA analyzer using BigDye Terminator v3.0 (Applied Biosystems). Sequence analysis was performed with the GCG software and the NCBI website [38]. Potential phosphorylation sites were detected using Scansite 2.0, with high stringency levels [21]. The sandflycyc sequence was submitted to the GenBank under the accession number DQ841151.

Quantitative RT-PCR

Firstly, mRNA was extracted with the QuickPrep™ Micro mRNA Purification kit (Amersham Biosciences) and reverse-transcribed with the TaqMan Reverse Transcription Reagents (Applied Biosystems) using the oligo-dT primer according to manufacturer instructions. Levels ofcyc mRNA relative to non-cycling levels ofrp49 were assayed by quantitative Real Time PCR using an ABI PRISM^® 7000 (Applied Biosystems) as previously described [14]. We used 3 different sets of primers forcyc and one forrp49.cyc primer pairs: 5'TGCCAAAACAATGCTTCCGG 3' & 5'ACGTTGCCCTTTGATCGACA 3'; 5'AATTGATGCCAAAACAATGC 3' & 5'AGAATCAACGTTGCCCTTTG 3'; 5'GATGCCAAAACAATGCTTCC 3' & 5'GTGCCCAGGACTTGAGGTAG 3'.rp49 primer pair: 5'CGATATGCCAAGCTAAAGCA 3' & 5'GGGCGATCTCAGCACAGTAT 3'. At least one of each primer in the pair spanned an exon/intron boundary to prevent amplification from any genomic DNA contamination. Indeed, melting-temperature curves showed a single amplified product and the absence of primer-dimer formation, which was confirmed by gel electrophoresis (data not shown). Non-template controls were included for each primer pair to check for any significant levels of contaminants. Standard curves were used to confirm that primers pairs had similar reaction efficiencies. Reactions were carried out in quadruplicates in a final reaction volume of 30 μl using 2× SYBR^® Green PCR Master Mix (Applied Biosystems) and primers at a final concentration of 500 nM. Amplifications were carried out for 50 cycles as follows: (i) 95°C, 10 sec; (ii) 60°C, 60 sec; (iii) 78°C, 30 sec (florescence recorded); (iv) repeat. Raw data were exported to EXCEL (Microsoft) for analysis.