Rhythmic expression of the cycle gene in a hematophagous insect vector

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]).

In D. melanogaster, CLK and CYC form a heterodimer that binds to upstream E-box sequences (CACGTG) in period (per) and timeless (tim), which in turn control their own expression by negatively regulating CLK/CYC mediated activation [1, 2]. In the second loop, the products of vrille (vri) and PAR domain protein 1 epsilon (Pdp1 ε), 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 [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 and D. melanogaster, there are some interesting differences in, for example, the transcriptional control of Clk and cyc expression. In the suprachiasmatic nuclei (SCN) of mammals (where the central pacemaker is located), Clk is constitutively expressed [5] and Bmal1 is rhythmic, reaching its maximum abundance at dawn [6, 7]. In contrast, cyc is constitutively expressed in D. melanogaster heads [8, 9], while Clk shows rhythmic expression on the mRNA level, peaking during the night-day transition (ZT 22-2) [10, 11]. Although the Drosophila 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 promoting per and tim expression [12, 13].

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 [14]. While per and tim cycle as in other insects, peaking around ZT 13 [15–17], Clk expression peaks around ZT 9–13, about half a day later than in D. melanogaster [10, 11, 14]. This difference in Clk expression is correlated with differences in locomotor activity. Drosophila shows a bimodal/diurnal pattern, whereas Lutzomyia is predominately unimodal/nocturnal [14]. 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 [14]. 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.

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 (Fig 2). 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 (except Drosophila) 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 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 [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 (Fig 4a). 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-boxes in 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 where cyc expression is also rhythmic (Fig 5a) [6, 7, 24]. Unlike most insects analyzed so far (where no oscillation of cyc mRNA was detected [9, 16, 23], but see Rubin et al [27]) sandfly cyc cycled robustly, beginning to rise at the end of the night (ZT 21) and peaking in the middle of the day ZT 5–9 (Fig 5a).

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 [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 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 [12, 13]. 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 [14]. 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 (ZT 13, when it can dimerize with the product of Clk and drive per and tim transcription [9, 14]).

In contrast to heads, cyc expression in female bodies was shown to be constitutive (Fig 5b). In Drosophila per was shown to be constitutively expressed in ovaries [22] causing a strong damping in per cycling in female bodies. In fact, sandfly per is also constitutive in female bodies [14]. The differential regulation of cyc through the sandfly body suggests that, as in Drosophila and mammals [29], 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 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, although per and tim expression are downregulated, Clk and cyc 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 diminished per and tim 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 activity in 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 in Drosophila do not appear to influence circadian behavior or molecular rhythms of per and tim [36].

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.