Expression of Clock Genes period and timeless in the Central Nervous System of the Mediterranean Flour Moth,Ephestia kuehniella

Abstract

Homologous circadian genes are found in all insect clocks, but their contribution to species-specific circadian timing systems differs. The aim of this study was to extend research within Lepidoptera to gain a better understanding of the molecular mechanism underlying circadian clock plasticity and evolution. The Mediterranean flour moth, Ephestia kuehniella (Pyralidae), represents a phylogenetically ancestral lepidopteran species. We have identified circadian rhythms in egg hatching, adult emergence, and adult locomotor activity. Cloning full-length complementary DNAs and further characterization confirmed one copy of period and timeless genes in both sexes. Both per and tim transcripts oscillate in their abundance in E. kuehniella heads under light-dark conditions. PER-like immunoreactivity (PER-lir) was observed in nuclei and cytoplasm of most neurons in the central brain, the ventral part of subesophageal complex, the neurohemal organs, the optic lobes, and eyes. PER-lir in photoreceptor nuclei oscillated during the day with maximal intensity in the light phase of the photoperiodic regime and lack of a signal in the middle of the dark phase. Expression patterns of per and tim messenger RNAs (mRNAs) were revealed in the identical location as the PER-lir was detected. In the photoreceptors, a daily rhythm in the intensity of expression of both per mRNA and tim mRNA was found. These findings suggest E. kuehniella as a potential lepidopteran model for circadian studies.

Keywords

circadian clock activity rhythms eclosion rhythm PER-like immunoreactivity Lepidoptera

In insects, circadian clocks control daily rhythms in various life processes. The molecular mechanism of the Drosophila clock model involves a transcription/translation negative feedback loop based on endogenous, approximately 24-h oscillations of period (per) and timeless (tim) transcripts and their corresponding proteins. When PERIOD (PER) and TIMELESS (TIM) proteins reach critical concentrations in the cytoplasm during the late evening, they translocate to the nucleus, where they block their own transcription. Within a population of ~150 brain cells coexpressing PER and TIM, small ventral lateral neurons, large ventral lateral neurons, and dorsal lateral neurons are important as circadian pacemakers for locomotor rhythms and pupal eclosion, while dorsal neurons (in subgroups DN1, DN2, and DN3) and glial cells may play more subtle roles in controlling circadian rhythmicity in Drosophila melanogaster (reviewed in Helfrich-Forster, 2005; Nitabach and Taghert, 2008).

To date, per and tim, as well as other clock genes, have been sequenced from various insect orders. Although homologous circadian genes have been found in most insect species, some groups, such as hymenoptera, lack the tim gene (Rubin et al., 2006; Ingram et al., 2012). Detailed neuroanatomical studies of circadian proteins within drosophilids (Hermann et al., 2013) further indicate that homologous factors may function differently in closely related species within a single genus.

In the order Lepidoptera, the circadian clock mechanism has been examined in detail in 4 species: the Chinese oak silkmoth, Antheraea pernyi; the monarch butterfly, Danaus plexippus; the silkmoth, Bombyx mori; and the hawkmoth, Manduca sexta. Sauman and Reppert (1996) described 2 distinct systems of PER regulation in A. pernyi. In the eye, PER regulation works in a manner similar to that found in Drosophila, but in the central brain, Apper messenger RNA (mRNA) and ApPER abundances oscillate synchronously, which is in contrast to the Drosophila model, in which a peak in Dmper mRNA is followed by a 4- to 6-h delayed peak in DmPER protein levels. This delay is a regulated process and is essential for timely PER-TIM nuclear translocation (Saez and Young, 1996; Price et al., 1998). ApPER and ApTIM proteins are coexpressed only in 4 large Ia₁ neurosecretory cells in each hemisphere in the dorsolateral protocerebrum (pars lateralis [PL]), which represents a significant reduction compared to dozens of PER/TIM-positive neurons and hundreds of PER-positive glia cells in the Drosophila brain. Both ApPER and ApTIM are restricted exclusively to the cytoplasm with no temporal movement into the nucleus, a critical feature of the Drosophila model.

An alternative circadian clock mechanism to the Drosophila model was proposed for the monarch butterfly (Zhu et al., 2008). Although per and tim genes are present in D. plexippus, the monarch employs Drosophila-like cryptochrome (cry1) and mammalian-like cryptochrome (cry2) genes as the key players in the central clockwork. The CRYPTOCHROME 1 (CRY1) functions as a blue-light photoreceptor for photic entrainment, whereas CRY2 functions as the major transcriptional repressor of the core transcriptional feedback loop. At the same time, Dpper and Dptim mRNA levels exhibit daily rhythms of similar phase to DpPER/DpTIM proteins in monarch heads, as in A. pernyi. DpPER and DpTIM are coexpressed in 2 neurons in each PL of the dorsolateral protocerebrum. Positive signals are exclusively cytoplasmic and do cycle in a light/dark regime (LD; Sauman et al., 2005; Zhu et al., 2008). However, functional analyses of relevant circadian genes in monarch cell lines revealed that TIM and PER play only auxiliary roles in the monarch CRY2-centric clock mechanism (Zhu et al., 2008).

In the head of the silkmoth, B. mori, Bmper and Bmtim transcripts and BmPER protein (Sehadova et al., 2004) show a rhythmic expression as in the A. pernyi brain. An exclusively cytoplasmic PER-like signal with daily oscillation was detected in 4 large neurons and, occasionally, in adjacent 2 to 4 small cells in each PL of the dorsolateral protocerebrum (Sehadova et al., 2004).

Four large PER-positive cells were identified in PL in the M. sexta brain. However, the PER staining was found not only in cytoplasm but also in nuclei of these cells with no evidence of PER oscillation in either cell compartment (Wise et al., 2002).

TIM coexpression with PER was not examined in B. mori or M. sexta. However, in both species, the 4 large cells in PL were distinguished as type Ia₁ neurosecretory cells according to their size, position, and coexpression of other circadian clock proteins or neuropeptides (Wise et al., 2002; Sehadova et al., 2004).

It is worth noting that the distribution of per gene products in B. mori and M. sexta, as well as both PER and TIM expression in D. plexippus, is more widespread in the brain-subesophageal complexes, eyes, and optic lobes. But these PER- or TIM-positive neurons and glial cells, other than Ia₁ neurosecretory cells, are usually missing the required characteristics for clock cells such as PER-TIM coexpression, PER and TIM daily oscillations in abundance that persist in constant darkness, or coexpression with other relevant circadian clock proteins (Wise et al., 2002; Sehadova et al., 2004; Sauman et al., 2005; Zhu et al., 2008).

We aim to enhance the understanding of the evolutionary plasticity of genes per and tim in the lepidopteran core clock mechanism. The Mediterranean flour moth, Ephestia kuehniella (Pyralidae, clade Obtectomera), represents a phylogenetically less divergent lepidopteran species compared to the other lepidopterans (clade Macrolepidoptera) that are under molecular investigation of the circadian clock. Our previous observations indicate that the locomotor activity of Ephestia males has a clear circadian character (Zavodska et al., 2012).

The main objectives of this study are to document circadian phenotypes of this species and characterize its 2 canonical circadian clock genes, period and timeless. To accomplish the latter goal, we employed cloning and sequencing techniques together with Southern and Northern blot analyses. In addition, in situ hybridization and immunocytochemistry were used to localize per/PER- and tim-expressing neurons in the central nervous system of the flour moth.

Materials and Methods

Insects

Wild-type (wt-c) and white eye (wa; Marec and Shvedov, 1990) strains of E. kuehniella from the Institute of Entomology were used for behavioral and molecular studies (see supplementary online materials [SOM] for details). Insect cultures were reared on milled wheat grains supplemented with a small amount of dried yeast under a 12-h light/12-h dark (12:12 LD) photoperiod at 21°C.

Activity Monitoring

Newly eclosed males (wa strain) were placed individually into glass tubes (diameter 25 mm, length 245 mm), and their locomotor activity was automatically recorded (Large Activity Monitors [LAM], Trikinetics, Waltham, MA) at a constant temperature of 25°C for 2 days in LD 12:12 followed by constant darkness (DD). Analyses were performed using MATLAB-based Flytoolbox (Levine et al., 2002).

Adult Ecdysis

E. kuehniella larvae were grown in a 12:12 LD regime until pupation. Three- to 5-day-old pupae were removed from cocoons and placed individually into glass vials (diameter 25 mm, flat bottom, length 80 mm). Vials were oriented vertically in LAM 25 monitor (TriKinetics, Waltham, MA) with pupae approximately 1 to 3 mm below the infrared beams. Emergence of adults was then recognized as the first signal from the sensor (interruption of the beam). Monitors with pupae were transferred to either a long photoperiod of 18:6 LD or remained in a 12:12 LD regime, at a constant temperature of 25°C. Sex was determined in the adult stage after eclosion.

Cloning the Clock Genes

Adult moths (wa strain) were rapidly frozen at −80°C and decapitated on dry ice. Total RNA was isolated from 20 to 30 adult E. kuehniella heads using RNA Blue (TopBio, Prague, Czech Republic). Complementary DNA (cDNA) was synthesized from 4 to 5 µg of total RNA using an oligo dT₍₁₆₎ primer and First Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Piscataway, NJ) or SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA). Full-length period (per) and timeless (tim) sequences were obtained using PCR with degenerate primers, primer walking strategy, and 3′, 5′RACE (3′ and 5′RACE System for Rapid Amplification of cDNA Ends, version 2; Invitrogen) (for details, see the Suppl. Tables S1-S3).

Quantitative PCR Analysis

The newly eclosed adults (wa strain) were entrained in the following illumination regimes: 21°C in either constant light (LL) for 6 days or 12:12 LD for 6 days followed by 1 day in constant darkness (DD). Adult E. kuehniella were collected every 4 h during the sixth day in LL or during the sixth day in LD and the following day in DD. Flour moths were rapidly frozen at −80°C at each time point and decapitated on dry ice. Heads from 20 adults were pooled to obtain one sample. Total RNA was isolated using TriReagent (Molecular Research Center, Cincinnati, OH). cDNA synthesis and quantitative PCR (qPCR) were performed as in Kobelkova et al. (2010); details on primers and conditions are included in the SOM (Suppl. Tables S4-S5).

Immunocytochemistry

Dissected heads were immediately fixed in modified Bouin-Hollande solution (supplemented with 0.7% mercuric chloride) overnight at 4°C. Heads were transferred through an ethanol series to chloroform and embedded in paraplast. Sections 4 to 10 µm thick were attached to microscopic slides. After deparaffinization in xylene and following rehydration, the sections were treated with Lugol’s iodine followed by 7.5% sodium thiosulfate to remove residual heavy metal ions and then washed in distilled water and phosphate-buffered saline supplemented with 0.3% Tween-20 (PBST). Blocking with normal goat serum (10% in PBST, 30 min at room temperature [RT]) was followed by incubation (overnight at 4°C in a humidified chamber) with a primary antibody diluted (1:200) in PBST. After rinsing with PBST (3 times for 10 min at RT), samples were incubated (1 h at RT) with a secondary antibody (in a 1:1000 solution of goat–anti-rabbit IgG conjugated to horseradish peroxidase [HRP]; Jackson ImmunoResearch, West Grove, PA) in PBST. Slides were then washed in PBST (3 times for 10 min at RT) followed by a final wash in 0.05 M Tris-HCl (pH 7.4; 10 min at RT). The enzymatic activity of HRP was visualized with hydrogen peroxide (0.005%) substrate and 3, 3′-diaminobenzidine tetrahydrochloride (0.25 mM in 0.05 M Tris-HCl, pH 7.4) as a chromogen. Stained sections were dehydrated, mounted in DPX mounting medium, and examined under a Zeiss Axioplane 2 microscope (Carl Zeiss, Oberkochen, Germany) equipped with Nomarski (DIC) optics and a CCD camera.

The polyclonal rabbit antibody used in this study had been raised against the PER S-region (KSSTETPLSYNQLN) of A. pernyi (Sauman and Reppert, 1996). The sequence is perfectly conserved in EkPER.

To verify that the secondary antibody alone did not recognize an antigen in our preparations, we replaced the primary antibody with normal goat serum. No significant staining above background was observed.

Immunostaining Quantification

Daily variations in the number of labeled cells and the staining intensity were examined in serial sections of the brain, the subesophageal ganglion (SOG), and the neurohemal organs (corpora allata and corpora cardiaca). Experimental insects were entrained for 5 to 7 days under the 12:12 LD photoperiod. The time was measured from the lights-on point that is referred to as 0 zeitgeber time (ZT) in animals exposed to a photoperiod. All investigations were carried out in 9 animals and included both sexes. The intensity of the immunostaining was assessed in 3 sets of preparations, each containing 3 series of heads dissected at ZT 0, 4, 8, 12, 16, and 20.

All preparations of every series were processed simultaneously. The length of exposure to DAB was set in preliminary runs with preparations from animals sacrificed at ZT 4. The staining with DAB was checked after the first 10 min and then every 5 min in 0.05 M Tris-HCl (pH 7.5), and the interval needed and sufficient for maximal staining was taken as the standard staining time (30 min).

The immunoreactivity was initially quantified subjectively with a 4-point scale, ranging from no reactivity (−) to weak (+), distinct (++), and strong reactivity (+++). Resulting staining was examined independently by 2 researchers first in randomized samples and then by comparing preparations from different circadian times of individual time series.

In Situ Hybridization

The freshly eclosed adults (wa strain) were entrained in 12:12 LD for 6 days. The moths were decapitated every 4 h during the sixth day in Ringer solution (DEPC water). Dissected heads were fixed in 4% paraformaldehyde overnight at 4°C, dehydrated through an ascending ethanol series to chloroform, and embedded in paraplast overnight at 60°C. Paraplast-embedded tissues were sectioned (10 µm), attached to glass slides (Superfrost Ultra Plus slides; Fisher Scientific, Waltham, MA), deparaffinized in xylene, and rehydrated through a descending ethanol series to phosphate-buffered saline solution (PBS). In situ hybridization was carried out using mRNA locator kit (Ambion) according to the manufacturer’s instruction manual. Slides were prehybridized at 58°C for 3 to 4 h and hybridized at 58°C overnight. The same DIG-labeled RNA per and tim probes used for Northern blot analyses were used for in situ hybridization. DIG-labeled sense RNA probes were used in the control experiments. Immunohistochemical detection of hybridized probes was done using Fab fragments of sheep anti–digoxigenin antibody conjugated to alkaline phosphatase (AP; Boehringer and Mannheim, Germany; 1:500 dilution in PBST, overnight at 4°C). The AP activity was visualized with the BCIP/NBT substrate system (PerkinElmer, Waltham, MA). Staining reaction was stopped by a 5-min wash in distilled water. Slides were dehydrated through an ascending ethanol series to xylene, mounted in DPX mounting medium, and examined with a Zeiss Axioplane 2 microscope.

Results

Rhythmic Activity of E. kuehniella

Our previous study (Zavodska et al., 2012) described locomotor activity of wild-type E. kuehniella adults under 3 different photoperiods. Since eye pigments complicate subsequent molecular and immunohistochemical procedures, we have examined whether the eye mutant E. kuehniella strain possesses a functional circadian clock by characterizing 3 different rhythmic behaviors: larval hatching, adult eclosion, and adult locomotor activity.

While locomotor activity was examined in constant darkness (Figure 1A) and shows a clear endogenous rhythm, larval and adult emergence was examined only in LD regime(s). Larval hatching occurs mostly during the photophase (Figure 1B), with a peak in the first half of the day. Adult emergence is maximal during the end of the photophase and beginning of the scotophase in both short (12:12 LD) and long (18:6 LD) photoperiods (Figure 1C).

Figure 1.

Periodic phenotypes of Ephestia kuehniella. Locomotor activity of adult males of the wa strain (left) and the periodogram calculated from DD data (right). Eggs were split into 2 groups and placed in opposite regimes of 12:12 LD; hatching occurred on day 5 (B). Adult emergence under the equinox photoperiod (C, top) and the long photoperiod (C, bottom).

Characterization of period and timeless cDNA Sequences

The Ekper cDNA transcript encodes a protein of 919 amino acids. Conserved sites (per^S and per^L) and domains typical of Drosophila PER protein (PAS dimerization domain, cytoplasm localization domain, and nuclear translocation signal) were localized in the EkPER sequence. Unlike Drosophila, the TG repeat region is not present in the flour moth sequence (Suppl. Figures S1A, S2).

For interspecies comparison, the major region of sequence similarity (Ek/Ap = 70%) in the per gene is the PAS domain; otherwise, the 3′ end is very variable with low sequence homology. Computer searching of GenBank using the Blastx algorithm revealed that the PER protein has the highest homology score with B. mori (Lepidoptera), although the uncorrected pairwise sequence divergence is relatively high (56.4%). This observation is in accordance with the finding that per is a rapidly evolving gene with only 14% of the alignable amino acid (aa) residues remaining invariant across the 26 species of Lepidoptera (Regier et al., 1998).

The flour moth tim cDNA transcript encodes a protein of 1228 amino acids. Conserved domains typical of Drosophila TIM protein (dimerization domains PER1 and PER2, nuclear translocation signal) were also recognized in flour moth TIM (Suppl. Figures S1B, S3). In addition, a 32-aa region is well conserved in E. kuhniella, which supports the notion of the functional importance of this domain in D. melanogaster (Suppl. Figure S3). It has been shown that this 32-aa sequence is necessary for restoring wild-type rhythms in tim⁰¹ flies. Deletion of this domain lengthened the circadian period to 36 to 38 h in D. melanogaster (Ousley et al., 1998).

Southern and Northern Analysis of per and tim

Southern analyses confirmed the single copy of per and tim, respectively, in the E. kuehniella genome. Identical results were obtained for both male and female genomic DNA (Suppl. Figure S4).

Northern analyses revealed clear hybridization signals using antisense per and tim sequence-specific RNA DIG-labeled probes. The size of the per transcript was approximately 9 kb (Suppl. Figure S5). The higher band (~10 kb) probably represents a sterical conformation form of the per transcript. Despite the prolonged heating step (65°C for 30 min) before loading RNA on the denaturing formamide gel, we were not able to fully eliminate this RNA conformation form.

The size of the flour moth tim transcript detected with the antisense DIG probe was 5.4 kb (Suppl. Figure S5B), which roughly correlated with the cloned tim transcript. The higher 2 bands (6.5 kb and 8 kb, respectively) are considered conformation forms of the 5.4-kb tim transcript. In control experiments using per and tim sense RNA probes, no signal was detected (Suppl. Figure S5B).

per and tim mRNA Levels Oscillate in E. kuehniella Heads

We have performed qPCR to analyze per and tim expression at the transcriptional level under 12:12 LD, DD, and LL conditions. The quantitative analysis of samples collected at 4-h intervals (ZT 0 corresponds to lights-on) revealed clear daily oscillations in the abundance of both per and tim mRNAs in LD (Figure 2A). Both transcripts showed minimal levels at ZT 4 and maximal levels at ZT 16 (Figure 2A). Although a similar trend continued in DD, the differences between minimal and maximal expression levels were not statistically significant (Figure 2A). For both per and tim, the corresponding mRNA levels remain flat in constant light with relatively high levels of expression at all examined time points, comparable to the average values in LD (Figure 2B). In addition, males and females were examined separately, and no differences in per and tim oscillation under LD conditions were found between the 2 sexes (data not shown).

Figure 2.

Expression of circadian clock genes period and timeless in adult heads in 12:12 LD followed by constant dark (A) and in constant light (B) at 21°C. Analysis of variance followed by Tukey’s post hoc comparison showed significant differences in expression of per (solid line; p < 0.01) and tim (broken line; p < 0.01) in the LD-DD photoregime. Means flanked by different letters were statistically different at p = 0.05. No significant differences were found in expression of per (p > 0.05) and tim (p > 0.05) in constant light. Relative values were standardized to the RNA levels of the ribosomal protein 49 (rp49, syn. RpL32) and normalized to the sample with the highest expression. Values represent the mean of 3 independent experiments ± SD. Open and black bars represent photophase and scotophase, respectively. Shaded bar depicts subjective day during constant darkness.

PER-Like Immunoreactivity in the E. kuehniella Central Nervous System

To identify putative circadian clock cells, we examined PER-like expression in the E. kuehniella CNS. Both males and females were kept under the LD photoregime and sacrificed at 4-h intervals, starting at ZT 0. Using a polyclonal antibody against A. pernyi PER, we detected PER-like immunoreactivity (PER-lir) in the central brain, optic lobes (OL), SOG, eyes, and retrocerebral complex (Figure 3). The staining intensity of PER-lir was quantified as absent, weak, distinct, or strong (Table 1).

Figure 3.

Localization and intensity staining of PER-like positive cells in the brain-subesophageal complex, eyes, and retrocerebral glands of Ephestia kuehniella. Animals were taken for examination at 4-h intervals from the 12:12 LD photoperiodic regime. Zeitgeber time was counted from lights-on (ZT 0; i.e., 0 h). (A) The schematic diagrams illustrate the topography of PER-lir cells at ZT 4 and ZT 16. (B) Strong cytoplasmic staining in the neurosecretory cells in the PI. The staining of axons was undetectable during the light phase of the LD photoperiod but showed prominent intensity during the dark phase. (C) Nuclear and cytoplasmic PER-like positive signal in the frontal region of the SPr. (D) Hundreds of PER-like positive nuclei occurred during the entire LD photoperiod in the OL. At ZT 0 and ZT 4, the cytoplasmic staining of cells was visible (arrowheads indicate the nuclei of the cells; arrows indicate cytoplasmic staining). Nuclear immunostaining of the photoreceptors disappeared in the middle of the dark phase and reached its maximal intensity in the middle of the light phase of the photoperiodic regime. (E) PER-like positive cells in the labial neuromere of the SOG. The black arrows indicate cytoplasmic staining of the cells; the arrowheads indicate staining of the nuclei. (F) Immunopositive fibers in the CC and CA. (G) The prominent daily oscillation of PER-lir in the photoreceptor nuclei. During the light phase, strong staining was detected in the photoreceptors (with the maximum at ZT 8); at ZT 12, only weak staining occurred, and at ZT 16, it disappeared completely. Black arrows indicate the nuclei of the photoreceptors set up along the ommatidium rhabdom, and arrowheads indicate the nuclei of the basal photoreceptors. White arrowheads indicate photopigment that accumulates in the eyes during the day (ZT 4, ZT 8). PI = pars intercerebralis; SPr = superior protocerebrum; OL = optic lobe; SOG = subesophageal ganglion; me = medulla; la = lamina; R = retina; CC = corpora cardiaca; CA = corpora allata. Bar = 100 µm applies to all micrographs in the row.

Table 1.

Localization and the intensity of PER-like immunostaining in the brain, eye, subesophageal ganglion, and neurohemal organs of Ephestia kuehniella kept under LD (12:12) photoregime.

Localization	ZT 0	ZT 4	ZT 8	ZT 12	ZT 16	ZT 20
PI
Cell-l	+++	+++	+++	+++	+++	+++
Axons	+++	−	−	−	+++	+++
SPr
Medial	+++	+++	+++	+++	+++	+++
Lateral	+++	+++	+++	++	++	++
IP_r
Lateral	+++	+++	+++	++	++	++
OL	+++	+++	+++	++	++	++
Pho
Long	++	+++	+++	−	−	+
Basal	++	+++	+++	+	−	+
SOG
Lb	+++	+++	+++	++	++	++
CC
Fibers	+++	+++	+ ++	+++	+++	+++
CA
Fibers	+++	+++	+++	+++	+++	+++

Immunoreactivity was quantified as absent (−), weak (+), distinct (++), and strong (+++). No differences in distribution and intensity of PER-ir were revealed with respect to the sex. PI = pars intercerebralis; SPr = superior protocerebrum; IPr = inferior protocerebrum; OL = optic lobe; Pho = photoreceptors; SOG = subesophageal ganglion; Lb = labial neuromere; CC = corpora cardiaca; CA = corpora allata; l = large; long = longitudinal.

In the central brain, prominent PER-lir was detected in both nuclei and cytoplasm of hundreds of cells in the frontal and the dorsal part of the superior protocerebrum (SPr), in the lateral inferior protocerebrum (IPr) at the border with the OL, and in the pars intercerebralis (PI; Figure 3A-C). In the PI, a group of 4 to 5 large immunopositive neurons and their axonal projections in each brain hemisphere was detected (Figure 3B). Strong immunostaining of these neurons was localized exclusively to the cytoplasm with no evidence of a daily rhythm in the intensity of staining or nuclear translocation. In contrast, their neurites exhibited a clear daily cycle in staining intensity with undetectable labeling during the light phase of the LD photoperiod (ZT 4-12) and prominent signal during the dark phase (ZT 16, 20; Figure 3A-C; Table 1).

Hundreds of PER-like positive cells with prominently stained nuclei at all examined ZTs were seen in the optic lobes (Figure 3D, Table 1). The cytoplasmic staining of these cells was also visible at ZT 0 and persisted to ZT 4.

In the eye ommatidia, a clear rhythmic PER staining was detected in the nuclei of 8 photoreceptor cells (R1-R8) setting up along the rhabdom as well as in a basal photoreceptor cell (R9). PER-lir in photoreceptors oscillated during the day with the maximal intensity during the light phase of the photoperiodic regime (ZT 4, ZT 8; Table 1) and lack of a signal in the middle of the dark phase (ZT 16; Figure 3A,D,G, Table 1).

At all ZTs, strong and consistent PER-lir was detected in the nuclei and cytoplasm of cells located medially and laterally in the labial neuromere of the SOG (Figure 3E). Axonal projections in both the corpora cardiaca and corpora allata showed strong immunostaining at all examined time points (Figure 3F, Table 1).

No differences in the spatial localization and diurnal changes of the PER-lir were found between males and females.

In Situ Hybridization of per and tim mRNA in the Flour Moth CNS

We next examined the flour moth brain-retrocerebral complex by means of in situ hybridization to find out whether the cells containing the PER-like antigen, as determined by immunocytochemistry, also express the per transcript. For the in situ analysis, we used a specific digoxigenin-labeled RNA probe corresponding to a ~1.2-kb sequence of the E. kuehniella per cDNA (Suppl. Figure S1A). In situ hybridization revealed a nuclear and cytoplasmic signal in restricted populations of cells in the pars intercerebralis, the lateral and medial regions of the SPr, the lateral IPr, optic lobes, photoreceptors, and the SOG (Figure 4A,B). All these labeled cells were found in identical locations as the PER-lir cells. In situ hybridization analysis under LD conditions at 4-h intervals did not reveal any detectable oscillations in the intensity of per mRNA expression in the brain-SOG complex. In contrast, a clear daily rhythm in the intensity of nuclear per mRNA expression was found in the eye photoreceptors (Figure 4B). Weak signal was observed in the early light phase (ZT 0 and ZT 4), while the maximal expression of the per mRNA appeared in the late dark phase (ZT 16 and ZT 20; Figure 4B).

Figure 4.

In situ expression of per messenger RNA (mRNA) (A, B) and tim mRNA (C, D) in the Ephestia kuehniella adult brain and eyes. (A) Cytoplasmic and nuclear labeling by antisense per RNA probe in the brain, SOG, OL, and photoreceptors. Expression of per mRNA corresponds to the localization of PER-lir cells. There was no hybridization signal with the sense per probe in the brain-SOG complex. (B) The daily oscillation of per mRNA expression in the compound eye photoreceptors. Weak nuclear hybridization signal with the antisense per probe was detected in the early light phase (ZT 0 and ZT 4); maximal expression of per mRNA (indicated by arrowheads) occurred in the late dark phase (ZT 16 and ZT 20). (C) The daily oscillation of tim mRNA expression in the photoreceptors. Weak nuclear hybridization signal with the antisense tim probe was detected during the light phase of photoperiodic regime; strong expression of tim mRNA (arrowheads) occurred in the dark phase. (D) Hybridization signal with the antisense tim probe in the PI, SPr, labial neuromere of the SOG, OL, and eye photoreceptors. Arrows indicate cytoplasmic labeling in the PI and in OL at ZT 16; arrowheads indicate nuclear expression of tim mRNA in OL at ZT 4. No hybridization signal with the sense tim probe was detected in the brain-SOG complex. PI = pars intercerebralis; SPr = superior protocerebrum; OL = optic lobe; SOG = subesophageal ganglion; me = medulla; la = lamina; CB = central body; R = retina. Bar = 100 µm (applies to all micrographs).

Similar to the per mRNA in situ expression pattern, hybridization signal with the antisense tim RNA probe corresponding to 1.4 kb of the tim cDNA sequence (Suppl. Figure S1B) was detected in many cells in the lateral SPr and medial SPr, labial part of the SOG, the optic lobes, and the photoreceptors. Clearly, cytoplasmic tim labeling was observed in the cells located in the pars intercerebralis (Figure 4D). Evidence of variation in tim cytoplasmic and tim nuclear distribution was detected in cells of the OL during the LD photoperiod: tim nuclear labeling occurred at ZT 4, while tim cytoplasmic labeling occurred at ZT 16 (Figure 4D). In the photoreceptors, a weak nuclear hybridization signal with the antisense tim RNA probe was detected during the light phase of the photoperiodic regime (ZT 4, ZT 8), while strong expression of tim RNA was observed in the dark phase (ZT 16, ZT 20; Figure 4C).

During the course of the per and tim in situ hybridization experiments, we employed the sense per and tim RNA probes as negative controls. No staining above background in the brain, optic lobes, or retrocerebral complex was detected (Figure 4A,D).

Discussion

Our previous work revealed daily rhythms in both female calling behavior and male locomotor activity of E. kuehniella. Female calling behavior showed a peak with maximum at the end of the scotophase, slowly declining during the photophase. Locomotor activity, measured in males, was bimodal, with a first peak after lights-off and a second peak at the end of the dark phase (Zavodska et al., 2012).

In the current study, we have identified 2 additional periodic events in the development of E. kuehniella: emergence of adults from pupa and hatching of larvae. Adult emergence occurs at the beginning of the scotophase, when more than 60% of adults appear during a 3-h window in both long and short photoperiods (Figure 1C). In contrast, larvae hatch from the egg shells during the photophase (Figure 1B). This behavior has a broader distribution, with a peak during the first 4 to 6 h of the photophase.

To further characterize the circadian clock components of E. kuehniella, we have cloned the full-length per and tim cDNAs. Ekper and Ektim possess all the important functional domains known from D. melanogaster and also conserved in lepidopteran species. Notable divergences in the per gene have been described among Lepidopterans. The Chinese oak silkmoth, A. pernyi, possesses 3 genomic per loci: per gene (Reppert et al., 1994), per-like gene, and a locus coding an endogenous antisense per RNA (Gotter et al., 1999). The antisense Apper RNA oscillates in antiphase to sense Apper RNA in the 8 neurosecretory cells in the central brain (Sauman and Reppert, 1996). The silkmoth, B. mori, carries only 1 copy of the per gene (Iwai et al., 2006), and per antisense RNA is expressed in the large cells, in both PI and PL as well as in the photoreceptor cells (Trang le et al., 2006). A single copy of the per gene was found in both sexes of the flour moth (Suppl. Figure S4A). Expression of antisense per RNA was not detected in the head of E. kuehniella, either by Northern blot (Suppl. Figure S5A) or by in situ hybridization (Fig. 4A). This suggests that the presence of antisense per RNA is not a universal trait of the lepidopteran circadian clock.

Both per and tim transcripts oscillate in abundance under 12:12 LD conditions, with the lowest expression at ZT 4 and maximum expression at ZT 16. The levels of per and tim transcripts remain at a constant and high level in continuous light (Figure 2B). Daily rhythms of per and tim mRNAs in LD with low levels during the day and high levels during the night were observed in heads of the silkmoths A. pernyi (Sauman and Reppert, 1996) and B. mori (Iwai et al, 2006), as well as in monarch butterfly brains (Zhu et al., 2008). In all 3 species, the rhythms of per and tim mRNAs persisted in DD.

In situ hybridization revealed daily cycles in intensity of per and tim mRNA expression in the nuclei of the eye photoreceptors (Figure 4B,C), while in the brain-SOG complex, oscillations in the intensity of mRNA expression were not detected. Weak signal in the photoreceptors was observed in the early light phase of the photoperiodic regime while the maximal expression of per and tim transcripts appeared during the dark phase (ZT 16 and ZT 20). In A. pernyi, per mRNA levels oscillated in photoreceptor nuclei with a similar phase: high mRNA levels occurred in the early dark phase and lowest mRNA levels in the middle of the light phase (Sauman and Reppert, 1996). In the hawkmoth, M. sexta, per mRNA was detected in the photoreceptor nuclei, but there was no evidence of a daily rhythm either in the number of photoreceptors expressing per or in the signal intensity (Wise et al., 2002). In E. kuehniella, many brain cells in the PI, the superior and inferior protocerebrum (Pr), OL, and SOG exhibited both cytoplasmic and nuclear per and tim mRNA expression (Figure 4). Obvious cytoplasmic per and tim mRNA labeling was observed in the cells located in the PI and the superior Pr, while nuclear distribution was detected predominantly in cells of the OL. We also observed variation in tim cytoplasmic and tim nuclear distribution during the LD photoperiod: tim nuclear labeling occurred at ZT 4, while tim cytoplasmic labeling occurred at ZT 16 (Figure 4D). Similar hybridization patterns, both in the intensity of per/tim mRNA labeling and in the location of cells expressing per and tim in the central brain-SOG complex, were observed at all ZTs under the LD photoregime. This observation suggests that the daily oscillation of mRNA levels during LD conditions (Figure 2) depends on rhythmic expression of per and tim in the photoreceptors of the compound eyes.

Daily changes in PER expression and its translocation from the cytoplasm to nucleus are regarded as a fundamental clock feature. This translocation was discovered in the brain of D. melanogaster (Liu et al., 1988; Siwicki et al., 1988) but could not be detected in the PER-expressing neurons of any Lepidoptera investigated so far. An overview of published results referring to PER-like staining patterns in neural tissues of 4 lepidopteran species—the hawkmoth, M. sexta; the silkmoth, B. mori; the Chinese oak silkmoth, A. pernyi; and the monarch butterfly, D. plexippus—is summarized in Table 2.

Table 2.

PER-lir staining patterns in the cephalic ganglia of lepidopteran species: the Mediterranean flour moth (Ephestia kuehniella), hawkmoth (Manduca sexta), silkmoth (Bombyx mori), Chinese oak silkmoth (Antheraea pernyi), and monarch butterfly (Danaus plexippus).

Lepidoptera Species	Numbers of PER-lir Cells and PER-lir Distribution					Daily Oscillation
Lepidoptera Species	CB	OL	SOG	Re	RcC	Cell Somata	Cell Axons
Ephestia kuehniella	4-5 PI (cyt)	dozens	dozens	Pho	Axons CC	Pho cells	PI cells
Pyralidae	Dozens PI, Pr	(cyt + nuc)	(cyt + nuc)	(nuc)	Axons CA
PYRALOIDEA	(cyt + nuc)
Bombyx mori	5-7 PI (cyt)	—	—	—	Axons CC	PL cells	SPrL cells
Bombycidae	4 + 2 SPrL (cyt)				Axons CA
BOMBYCOIDEA
Antheraea pernyi	8 SPrL (cyt)	—	—	Pho	Axons CC	PL cells	PL cells
Saturniidae				(nuc)	Axons CA	Pho cells
BOMBYCOIDEA
Manduca sexta	4 SPrL (cyt + nuc)	Dozens	—	Pho	Axons CC	—	—
Sphingidae	Dozens PI, Pr			(nuc)	Cells CA
SPHINGOIDEA	(nuc)
Danaus plexippus	8-12 PI (cyt)	8 (cyt)	—	—	Cells CC	PI cells, SPrL cells	SPrL cells
Nymphalidae	2 SPrL (cyt)
PAPILIONOIDEA

Numbers of cells are per one half of the respective ganglion. The data used in the table were published: Sehadova et al., 2004 (Bombyx mori); Sauman and Reppert, 1996 (Antheraea pernyi); Wise at al., 2002 (Manduca sexta); Sauman et al., 2005 (Danaus plexippus). PI = pars intercerebralis; SPrL = lateral superior protocerebrum; Pr = protocerebrum; PL = pars lateralis; OL = optic lobe; SOG = subesophageal ganglion; Re = retina; Pho = photoreceptors; RcC = retrocerebral complex; CC = corpora cardiaca; CA = corpora allata; cyt = cytoplasmic distribution; nuc = nuclear distribution; — = not detected.

In the cephalic ganglia of the flour moth E. kuehniella, we detected PER-like expression in both nuclei and cytoplasm of hundreds of cells in the frontal and the dorsal part of the SPr, IPr, PI, OL, SOG, and eyes (Figure 3, Table 1). A group of large Ia₁ neurosecretory cells with cytoplasmic PER staining was recognized in all lepidopteran species in the superior protocerebrum: either in the PI of E. kuehniella (4-5 neurons in each hemisphere) or in the PL of the silkmoth A. pernyi (Sauman and Reppert, 1996) and the hawkmoth M. sexta (4 neurosecretory cells in each hemisphere; Wise at al., 2002) or alternatively in both regions in the monarch butterfly D. plexippus (Sauman et al., 2005) and the silkmoth B. mori (Sehadova et al., 2004).

Daily oscillations in the intensity of PER staining were observed in the PL cells in B. mori and A. pernyi, in both PL and PI cells in D. plexippus, but not in the large PER-positive cells of E. kuehniella and M. sexta. In all these lepidopteran species, the levels of PER protein in the Ia₁ cells reached their maximum during the night (peak levels from ZT 8-16 in B. mori, ZT 16-22 in A. pernyi, and ZT 15-18 in D. plexippus) and were low (B. mori) or undetectable (A. pernyi, D. plexippus) during the early day of a light-dark photoperiod. Axons of corresponding PL cells in the silkmoths B. mori and A. pernyi and PI cells in the monarch D. plexippus also manifest daily changes in the intensity of PER signal with the same patterns of daily fluctuations of staining, as shown in the perikarya of these cells. Neurites of PI cells in the flour moth show daily oscillations in PER staining intensity despite the invariable PER signal in the cell perikarya (Figure 3, Table 1, and Suppl. Table S6). During the dark phase, there were clearly visible PER-positive processes, while during the light phase (between ZT 4 and ZT 12), staining in neurites was not detectable.

Hundreds of small neurons with positive nuclear PER labeling were observed in the superior protocerebrum of the flour moth and the hawkmoth. In the optic lobes, dozens of PER-positive nuclei occurred in the flour moth and the hawkmoth, while only a small group of cells with explicitly cytoplasmic staining was detected at the base of the optic lobes in the monarch butterfly (Table 2).

In the compound eyes of the flour moth E. kuehniella, the silkmoth A. pernyi, and the hawkmoth M. sexta, PER-lir was localized in the nuclei of the phororeceptors (Table 2). While no evidence of daily cycles in the intensity of PER staining was found in M. sexta photoreceptor cells (Wise et al., 2002), robust daily changes of PER levels were detected in the photoreceptors of E. kuehniella (Figure 3G, Suppl. Figure S1) and A. pernyi (Sauman et al., 1996). In the flour moth, the daily rhythm is characterized by intense staining in the eyes during the light phase (from ZT 4-8) and by low levels during the dark phase. No PER-lir was detected in the middle of the dark phase of LD (Figure 3G). Comparison of the PER-lir with the fluctuation of per mRNA levels in the nuclei (Figure 4B) indicates a delay of protein accumulation by 6 to 8 h. This temporal delay between per mRNA and PER protein rhythms as well as the nuclear daily fluctuation of PER in the E. kuehniella eyes corresponds to the principal attribute of the circadian clock mechanism.

PER staining in the lateral and ventral part of the SOG in the flour moth is unique among the studied lepidopteran species. PER-lir occurs in the neurohemal organs, both corpora allata and corpora cardiaca, at all investigated ZTs (Figure 3E, Table 1).

Our previous comparative study indicated that the number of PER neurons in the brain is significantly higher in phylogenetically more ancestral apterygote and exopterygote insects than in Holometabola (Zavodska et al., 2003). The Mediterranean flour moth E. kuehniella (Pyralidae) represents a less divergent lepidopteran species (clade Obtectomera), while B. mori, A. pernyi, M. sexta, and D. plexippus all belong to the phylogenetically more advanced clade Macrolepidoptera (Kristensen et al., 2007). From this phylogenetic perspective, E. kuehniella might represent a species with an ancestral lepidopteran clock. Notably, the number of PER-like positive cells varies remarkably within lepidopterans (Table 2): there are hundreds of stained cells throughout the brain of Ephestia, while there are fewer than 20 PER-like positive cells in the macrolepidopteran species.

In this study, we have described 2 population rhythms, each with peaks at different times of day. It would be interesting to determine the underlying connection of circadian clocks and hormonal regulation of these particular developmental events. Periodic eclosion of D. melanogaster served as an elegant selection method in screening for circadian mutants (Konopka and Benzer, 1971). Because of easy rearing in laboratory conditions and a reasonably short life cycle, E. kuehniella became established as a genetic model for Lepidoptera using available classical forward genetic tools (Marec et al., 1999). The recent revolution of genome editing approaches, together with progress in next-generation sequencing, brings reverse genetic tools to nearly all organisms. Hence, E. kuehniella might be established as a practical model for lepidopteran molecular chronobiology.

Footnotes

Acknowledgements

We thank Marie Korchová for help with rearing insects, František Marec for continuous support in Ephestia research, and 2 anonymous reviewers for useful suggestions. This work was funded by the Grant Agency of the Czech Republic project 14-32654J to D.D. and by Research Organization Development (RVO) to R.Z. We acknowledge the use of research equipment funded from the European Union program FP7/2007e2013, grant 316304.

Conflict of Interest Statement

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Notes

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