|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online February 27, 2009
Journal of Experimental Biology 212, 867-877 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.027003
Roles of PER immunoreactive neurons in circadian rhythms and photoperiodism in the blow fly, Protophormia terraenovae
Department of Biology and Geosciences, Graduate School of Science, Osaka City University, Sumiyoshi, Osaka 558-8585, Japan
* Author for correspondence (e-mail: shigask{at}sci.osaka-cu.ac.jp)
Accepted 24 December 2008
 |
Summary |
|---|
 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Key words: activity rhythm, diapause, lateral neurons, pigment-dispersing factor, photoperiodic response
|
INTRODUCTION |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
; Nakao et al.,
2008; Turck et al.,
2008), less knowledge is available in the case of insects. Insect
photoperiodism has three physiological components: the photoreceptor, the
photoperiodic clock and the effecter
(Saunders, 2002). The
photoperiodic clock entails a time-measurement system that usually measures
the length of darkness per day (Saunders,
2002).
There have been several investigations of the physiological mechanisms
underlying the photoperiodic clock in insects
(Vaz Nunes and Saunders, 1999;
Saunders, 2002), and it is
generally accepted that circadian oscillators are involved in the
time-measurement system (Vaz Nunes and
Saunders, 1999; Veerman,
2001). To elucidate molecular or neural mechanisms underlying the
photoperiodic clock, an understanding of circadian oscillator genes and
neurons, and their relations with photoperiodism is needed. Based on the
extensive knowledge of the circadian clock mechanism at the molecular level in
Drosophila melanogaster
(Stanewsky, 2003), several
studies have tested the hypothesis that circadian clock genes function in the
photoperiodic clock.
Two circadian clock genes have been examined for their roles in
photoperiodism. Adult female D. melanogaster that have arrhythmic
mutant alleles of the circadian clock gene period
(per0 mutants) show photoperiodic control of reproductive
diapause, but the critical day length is less than in wild-type flies. This
suggests that per is not causally involved
(Saunders et al., 1989). In
another drosophilid fly, Chymomyza costata, the circadian clock gene
timeless has been shown to be crucial for photoperiodic control of
larval diapause. In a non-photoperiodic-diapause (NPD) strain of C.
costata, a single autosomal gene locus encoding tim was mutated,
and both circadian eclosion rhythms and photoperiodic control of larval
diapause were lost (Pavelka et al.,
2003). The mutant lacking tim still enters diapause when
exposed to low temperatures, suggesting that tim plays a role not in
diapause induction, but in photoperiodic mechanisms
(Riihimaa and Kimura, 1989).
Recent studies on D. melanogaster have suggested that tim
directly affects the incidence of diapause through circadian photoreception
(Tauber et al., 2007;
Sandrelli et al., 2007).
Although circadian clock genes have been the focus of studies on the
relationship between the circadian clock and photoperiodic mechanisms,
investigations of circadian clock neurons are also important. Insects have
multiple circadian oscillator systems, and circadian clock genes are expressed
in many cells in the whole body. In D. melanogaster, expression of
per has been reported to oscillate throughout the body, and
per in different tissues appears to drive different rhythms for
various physiological phenomena (Plautz et
al., 1997). Previous studies have shown the importance of the
brain in photoperiodic mechanisms (Bowen et
al., 1984); therefore, neurons expressing circadian clock genes in
the brain should be investigated to elucidate photoperiodic mechanisms.
Since Helfrich-Förster
(Helfrich-Förster, 1995)
first characterized clock-gene-expressing neurons in the brain, understanding
of circadian oscillator mechanisms that result in complex behavioural rhythms
has progressed in D. melanogaster
(Stoleru et al., 2004;
Grima et al., 2004;
Rieger et al., 2006). In the
D. melanogaster brain, six groups of neurons that express a set of
circadian clock genes have been identified, and each neuron group appears to
have a different role in behavioural rhythms
(Helfrich-Förster et al.,
2007; Grima et al.,
2004; Stoleru et al.,
2004; Rieger et al.,
2006). Although in D. melanogaster circadian clock genes
and neurons are well known, photoperiodic responses in this species are very
shallow and difficult to assay. Clock-gene-expressing neurons or their
protein-immunoreactive neurons have been examined in many species
(Frisch et al., 1996;
Sauman and Reppert, 1996;
Závodská et al.,
2005; Codd et al.,
2007); however, their roles in behavioural rhythms or
photoperiodism have not been identified. Only in the hawk moth Manduca
sexta, has the loss of photoperiodic control of pupal diapause been shown
after ablation of per-expressing neurons
(Wise et al., 2002;
Shiga et al., 2003). However,
it is not known whether these neurons have roles in circadian rhythm
oscillations in this species. Determining whether circadian clock neurons are
a component for the photoperiodic clock would help identify photoperiodic
clock neural networks and reveal integration mechanisms of photoperiodic
information that may be active in the development of seasonal phenotypes.
Adult female blow fly Protophormia terraenovae (Robineau-Desvoidy
1830) (Diptera, Calliphoridae) show photoperiodic control of reproductive
diapause. Females reproduce under long-day conditions, and enter diapause
under short-day conditions (Numata and
Shiga, 1995). The present study examined circadian clock neurons
in the brain of P. terraenovae, and the effect of ablation of these
neurons on photoperiodism. The results indicate that small ventral lateral
neurons (s-LNvs), which are immunoreactive to both Period (PER) and
a neuropeptide, the pigment-dispersing factor (PDF), are prerequisites for
circadian rhythm activity as in D. melanogaster. Furthermore,
ablation of the s-LNvs region resulted in a loss of photoperiodic
discrimination. Involvement of s-LNvs in photoperiodism and a
plausible neural network for photoperiodic control of diapause are
discussed.
|
MATERIALS AND METHODS |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
). For PER immunocytochemistry analysis and for recording
activity rhythms, insects were reared under 12 h:12 h L:D at
20±1°C. On days 6–11 (6 to 11 days after adult emergence),
adult females were used for immunocytochemical analysis. For the
photoperiodism experiments, insects were reared under 12 h:12 h L:D at
25°C. Ten to 15 females were collected on day 0, and held under 18 h:6 h
L:D or 12 h:12 h L:D at 25°C. During days 1–14, the females were fed
sucrose and water. Beef liver, which is necessary for ovarian development, was
given for the last 3 days, and ovarian development was examined on day 14.
Ovarian stages were determined according to Matsuo et al.
(Matsuo et al., 1997). Flies
with previtellogenic ovaries at stages 1 and 2– were considered in
diapause, whereas those with vitellogenic ovaries at stages 2+ to 6 were
deemed reproductive (i.e. non-diapause).
Immunocytochemistry
The heads of female flies were cut off at zeitgeber time 0–1 (ZT
0–1; 0–1 h after light-on) and the posterior cuticle removed to
expose the brain to fixatives. The head was fixed in 4% paraformaldehyde for 4
h at 4°C for whole-mount preparations, or in aqueous Bouin's fixative
overnight at room temperature for paraffin sections. PER immunocytochemistry
was performed using the ABC method (Vectastain ABC standard kit; Vector
Laboratories, Burlingame, CA, USA).
For staining of brain whole mounts, the brain was excised and washed in phosphate-buffered saline (PBS) with 0.5% Triton X-100 (PBST) overnight with several changes of PBST. The brain was incubated in 0.3% H2O2 for 1 h at room temperature to reduce endogenous peroxidase activity and in 0.5% BSA for 1 h. Then, goat anti-D. melanogaster-PER antiserum (sc-15720; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added at a working dilution of 1:1000. This polyclonal antibody was raised against a peptide mapping at the N terminus of PER of D. melanogaster origin. Brains were kept in the primary goat antiserum for 3 days at 4°C. This was followed by incubation in a secondary antiserum, donkey anti-goat immunoglobulin conjugated with biotin (705-065-003; Jackson ImmunoResearch Laboratories, West Grove, PA, USA), at a dilution of 1:200 for 1 day at 4°C. Both primary and secondary antisera were diluted in PBST containing 0.5% bovine serum albumin. Whole brains were incubated in an avidin–biotin complex solution at a dilution of 1:100 for 1 day at 4°C. After washing in PBST, the brains were preincubated in diaminobenzidine (Sigma, St Louis, MO, USA) for 1 h at 4°C, and incubated in a mixture of 0.01% H2O2 and 0.03% diaminobenzidine for 15–30 min at room temperature. After washing with PBST, whole-mount preparations were dehydrated in an ethanol series, and cleared in methyl salicylate for observation.
Double labelling with anti-PER antiserum (ABC method) and anti-PDF
antiserum (fluorescence immunocytochemistry) was performed on paraffin
sections. Paraffin sections (8 µm thick) were made using a standard
protocol. After thoroughly removing the paraffin, sections were incubated in
0.3% H2O2 for 30 min at room temperature. Subsequently,
they were incubated in PBS with 0.5% BSA for 20 min at room temperature and
then in the primary PER antiserum (1:1000) overnight at 4°C. After washing
with PBS, the primary antiserum-treated sections were incubated in the
secondary antiserum conjugated with biotin for 1 h, and then in an
avidin–biotin complex solution for 1 h at room temperature. After
washing with PBS, sections were incubated in a mixture of 0.01%
H2O2 and 0.03% diaminobenzidine for 2–7 min at
room temperature. Then, sections were washed thoroughly in PBS, and the
PER-stained sections were processed for PDF immunocytochemistry. The rabbit
anti-Gryllus bimaculatus-PDF antiserum was provided by Dr K. Tomioka
(Okayama University, Okayama, Japan). The epitope structures recognized by the
anti-Gryllus bimaculatus-PDF have been well characterized by
enzyme-linked immunosorbent assay (Honda
et al., 2006). The sections were incubated in the anti-PDF
antiserum at a dilution of 1:5000 for 1 day at 4°C. Swine anti-rabbit
immunoglobulin antiserum conjugated with tetramethylrhodamine isomer R (R0156;
Dako, Glostrup, Denmark) was used as a secondary antiserum at a dilution of
1:200 for 1 day at 4°C. After washing thoroughly in PBS, sections were
dehydrated and mounted in methyl salicylate.
A specificity test was performed using a pre-adsorption technique. The anti-PER antiserum at a dilution of 1:1000 was incubated in 200 µgml–1 of Period protein (sc-15720 P; Santa Cruz Biotechnology) overnight at 4°C. Immunocytochemistry was performed on whole-mount preparations using the antigen–antiserum complex instead of the primary antiserum. No staining was observed in this control experiment (data not shown).
In brains subjected to surgical operation, PDF immunocytochemistry was undertaken after recording activity rhythms or ovarian stages. Whole-mount brains were processed for PDF immunocytochemistry using the above mentioned ABC method. A dilution of 1: 5000 of the rabbit anti-PDF antiserum was used as the primary antiserum.
Images were studied with a compound photomicroscope (BX50-33DIC, Olympus, Tokyo, Japan) or an epifluorescence microscope (BX50-34FLA-3; Olympus). Double-labelled neurons were viewed as a bright-field image for diaminobenzidine, and the exactly the same area at the same depth was viewed as a fluorescence image for tetramethylrhodamine. The images were digitalized with a CCD camera (CoolSNAP; Nippon Roper, Chiba, Japan) and processed using Adobe Photoshop 6.0 (Adobe System Incorporated, Tokyo, Japan) for colour adjustment to the whole image if necessary, and by Corel Draw 9.0 (Corel, Ottawa, ON, Canada) for lettering.
Surgical operations
Surgical removal of PDF-immunoreactive (PDF-ir) neurons in the optic lobe
was performed to examine their roles in circadian rhythms and photoperiodism.
For examination of their role in activity rhythms, regions including large and
small PDF somata were ablated bilaterally (site 2 in
Fig. 1A). Because rhythm
deficiency was detected when small PDF somata were lost, regions smaller than
those shown in site 2 (site 4 in Fig.
1B) were ablated for examination of their role in photoperiodism.
Day-1 females were mounted in clay with the frontal face exposed and then
placed on ice for 10–30 min. Subsequently, under a stereomicroscope a
small pool of 0.9% NaCl was placed on the fly's face and bilateral vertical
incisions were made along the medial edge of each compound eye with bilateral
horizontal incisions above the antennae. The frontal face of the head cuticle
was then opened to expose the anterior brain surface. For examination of role
of PDF-ir neurons in circadian activity rhythms, portions of the anterior base
of the medulla (site 2 for the test group) or of the anterior-lateral region
of the protocerebrum (site 1 for the control-operated group) were bilaterally
ablated with a sharpened tungsten needle under 0.9% NaCl solution
(Fig. 1A). Following the
operation, the cuticle was returned to its original position. In a sham
operation, females were processed as above; however, no brain portions were
removed. To examine the role of small PDF-ir neurons in photoperiodism,
smaller portions of site 4 were removed
(Fig. 1B). As a control, the
anterior dorsal part of the protocerebrum was bilaterally ablated (site 3
operation; Fig. 1). A
sham-operated group was also prepared.
|
Recording and analysis of locomotor activity rhythms
The activity recording system was adopted from Hamasaka et al.
(Hamasaka et al., 2002).
Locomotor activity was recorded as the number of times that the fly
interrupted the infrared beam (EE-SPW321, Omron, Kyoto, Japan). The number of
events, summed every 6 min, was collected by a Microsoft Windows-based
personal computer. Following surgical operation, adult females on day1 were
individually placed in the recording chamber. Activity was recorded under
constant darkness (DD) at 20°C for 7–8 days and subsequently under
12 h:12 h L:D or 18 h:6 h L:D at 25°C for 10 days. The light intensity
(1.4 W m–2) was provided by a white fluorescent lamp (FL15W;
National, Osaka, Japan) during the photophase. Rhythmicity was evaluated by a
2 periodogram (Sokolove
and Bushell, 1978). To analyse diel activity levels, relative
values of events every 30 min were calculated each day, and daily means of the
relative values were calculated for each fly. Average values of the daily
means were calculate, and were plotted as relative activity levels for each
experimental group (see Fig.
6).
|
|
RESULTS |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
). We
used an antibody against D. melanogaster PER in our experiments. In
whole-mount preparations, three cell clusters in the boundary between the
optic lobe and mid-brain, and two clusters in the protocerebrum were
distinctively labelled (Figs 2
and 3). After the nomenclature
used in D. melanogaster, cell clusters at the boundary were named
dorsal lateral neurons (LNds), large-type ventral lateral neurons
(l-LNvs) and small-type ventral lateral neurons (s-LNvs)
(Helfrich-Förster, 1995;
Kaneko and Hall, 2000). In the
dorsal protocerebrum, two groups of dorsal neurons (DNs) were found. We
designated a cluster in the medial region as medial dorsal neurons
(DNms) and a cluster in the lateral region as lateral dorsal
neurons (DNls). In comparison with D. melanogaster,
Protophormia DNms seem to correspond to Drosophila
DN1s and DN2s and Protophormia DNls
correspond to Drosophila DN3s.
|
|
Double labelling with anti-PER and anti-PDF antisera was carried out
(Fig. 4) because the cell
locations of l-LNv, s-LNv and DNm were quite
similar to that of PDF-ir somata in P. terraenovae
(Nässel et al., 1993;
Hamanaka et al., 2007). All
four l-LNvs and four of five s-LNvs were also
immunolabelled with PDF antiserum. Four large and four small PDF-ir neurons
with somata at the anterior base of the medulla were l-LNvs and
s-LNvs, respectively (Fig.
4A,B). In the posterior dorsal protocerebrum, DNms
appeared in close proximity to the PDF somata. There were eight PDF somata in
the pars lateralis. Of these, three are reported to extend axons into the
retrocerebral complex to innervate the corpus cardiacum and hypocerebral
ganglion, and five are local neurons
(Hamanaka et al., 2007). PER
immunoreactivity mainly occurred in different cells from PDF somata in the
pars lateralis. However, at least one cell seemed to be labelled by both PER
and PDF antisera (Fig. 4C). In
most sections, the PER antiserum labelled both the nuclear and cytoplasmic
regions, whereas the PDF antiserum mainly labelled cytoplasmic areas
(Fig. 4). Although the PDF
antiserum stained axons and fibres along with somata, the PER antiserum
stained only somata.
|
Effects of removal of PDF-immunoreactive neurons on circadian activity rhythm
Activity under DD was classified into three patterns: rhythmic, obscure and
arrhythmic, and their incidences were compared
(Fig. 5). In the obscure
pattern, activity was classified neither as rhythmic nor typically arrhythmic
under DD (Fig. 5D). Most intact
and all sham-operated flies free-ran with a period of 24.9±0.6 h (mean
± s.d., N=31) in intact and 24.9±0.9 h (N=14)
in sham-operated flies under DD (rhythmic pattern), and were entrained to
short-day and long-day cycles with activities in the photophase
(Fig. 5A,
Fig. 6). In both short- and
long-day cycles, activities continued during the photophase irrespective of
the photophase length (Fig.
6).
|
|
When the anterior base of the medulla was bilaterally ablated (N=31, site 2 operation) (Fig. 1A), 25.8% of the flies showed arrhythmicity and 29.0% showed the obscure pattern under DD (Fig. 5C,D, Fig. 8A). The remaining flies were rhythmic with a free-running period of 24.6±1.0 h (N=14) under DD. In the obscure pattern group, some females showed rhythmic activity for a few days immediately after surgery, followed by arrhythmia (Fig. 5D). The rhythmic, obscure, or arrhythmic activities observed under DD continued under LD conditions in most females. Some females showing rhythmic patterns under DD became arrhythmic under LD. No females with the arrhythmic pattern under DD were subsequently rhythmic under LD. After the site 2 operation, masking effects, in which activities in the scotophase are higher than those in the photophase, were observed in some flies both under long- or short-day LD cycles (Fig. 5D, Fig. 6). The masking effects under LD conditions occurred irrespective of the activity patterns under DD. In the site 2-operated group, average activity levels in the scotophase were almost equal to or higher than those in the photophase (Fig. 6).
|
Fig. 7 shows tracings of PDF-ir neurons in the intact and site 2-operated groups. After the site 2 operation, different numbers of PDF-ir neurons in the optic lobe remained. The number of remaining PDF somata was counted in each fly, and the flies were then grouped according to these numbers, and incidences of the three activity patterns were calculated for each group (Fig. 8B). When flies were sorted according to the number of large PDF somata, no correlations were found between incidence of the rhythmic pattern and the number of large PDF somata (Fig. 8B, left). However, when the same data were sorted according to the number of small PDF somata, rhythmic pattern incidences depended on the number of somata (Fig. 8B, right). The data show that the fewer small PDF-ir neurons that remained, the fewer flies that showed the rhythmic pattern. In females from which all large PDF somata were removed (N=7; Fig. 8B, left), two, three and two females showed arrhythmic, obscure and rhythmic pattern, respectively. Both of the arrhythmic females had no small PDF somata. The numbers of small PDF somata in the three females exhibiting the obscure pattern were four, zero and zero. The number of small PDF somata in the two females showing rhythmic activity was one and seven. Even in the complete absence of large PDF somata, rhythmic activity occurred, if some small PDF somata were present.
Effects of removal of PDF-immunoreactive neurons on photoperiodism
The activity pattern results suggested that small PDF-ir neurons are
important for generating an activity rhythm under DD. Therefore, brain regions
containing small PDF somata were bilaterally ablated to examine the effects on
photoperiodism (site 4 operation; Fig.
1B). As a control, the anterior dorsal boundary between the optic
lobe and mid-brain was bilaterally ablated (site 3 operation;
Fig. 1B). In the intact,
sham-operated and control-operated groups, most flies entered diapause under
short-day conditions, whereas under long-day conditions most flies were
reproductive (Fig. 9). Diapause
incidences in intact, sham-operated, and control-operated groups under
short-days were significantly higher than those under long-days
(P<0.05, Tukey type multiple comparison for proportions)
(Zar, 1999)
(Fig. 9A). In the site
4-operated group, diapause incidence was 55.1% (N=49) under short-day
and 48.4% (N=31) under long-day conditions, and no significant
difference was detected (P>0.05, Tukey type multiple comparison
for proportions; Fig. 9A).
|
After examination of ovarian stages, brains were subjected to PDF immunocytochemistry. In the control-operated group (site 3 operation) PDF-ir neurons were successfully stained in all females (N=12) under short-day conditions and in 13 of 14 females under long-day conditions, whereas in the site 4-operated group, 46 of 49 females under short-day and 30 of 31 females under long-day conditions showed successful staining. In the control-operated group, all PDF somata (four pairs of large somata and four pairs of small somata) remained in most females. In most site 4-operated females, half or more of the large PDF somata remained, and half or more of the small PDF somata were ablated. No correlations were detected between the remaining number of large PDF somata and diapause phenotypes (data not shown). This was also the case for the number of small PDF somata (Fig. 9B). Even in females in which all the small PDF somata were ablated, both diapause and non-diapause females were observed (Fig. 9B). In those females with no small PDF somata remaining, under short-day conditions the number of large PDF somata was two to eight in both diapause (N=10) and non-diapause females (N=9), and under long-day conditions there were five to eight large PDF somata in diapause (N=6) and four to eight in non-diapause females (N=3). Although no significant correlations between the remaining number of PDF somata and diapause incidences were detected after site 4 operations, the effect of ablation of site 4 was different from that of site 3. This suggests that site 4 is important for photoperiodic control of diapause.
|
DISCUSSION |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
; Baylies et al.,
1987), and circadian oscillation of per mRNA or
distribution of per-expressing neurons has been shown in different
insect orders (Frisch et al.,
1996; Sauman and Reppert,
1996; Goto and Denlinger,
2002; Wise et al.,
2002; Hodková et al.,
2003; Závodská
et al., 2005; Iwai et al.,
2006; Codd et al.,
2007; Moriyama et al.,
2008). However, few studies have demonstrated that per or
per-expressing neurons are functionally important for circadian
behavioural rhythms or photoperiodism. In G. bimaculatus, it has been
reported that per knockdown by RNAi suppresses circadian rhythms in
locomotor activities and electrical activities of optic lobe neurons
(Moriyama et al., 2008). In
M. sexta, surgical ablation of per expressing neurons from
larval brains caused loss of photoperiodic control of pupal diapause
(Wise et al., 2002;
Shiga et al., 2003). Although
these studies suggest the importance of per or
per-expressing neurons in clock mechanisms, there have been no
reports on how these neurons integrate or process timing information in the
brain. The present study detected five groups of PER-ir neurons in the brain
of P. terraenovae and the results suggest that PER-ir
s-LNvs are important for driving circadian activity rhythms and the
site 4 containing s-LNvs for photoperiodism. These results,
together with the results of our previous studies, suggest a plausible neural
network for photoperiodic control of diapause.
Distribution of PER-immunoreactive neurons
In P. terraenovae, distribution of PER-ir cells in the brain and
colocalization patterns of PER with PDF were similar to that reported in
D. melanogaster
(Helfrich-Förster, 2003).
Also five s-LNvs, four of which were PDF-immunopositive were shown
to be present in P. terraenovae as in D. melanogaster. Only
one PDF-immunonegative s-LNv is present in P. terraenovae
and D. melanogaster, whereas four PDF-immunonegative
s-LNvs were found in the house fly Musca domestica
(Codd et al., 2007). However,
the numbers of l-LNvs and LNds were similar in D.
melanogaster, M. domestica and P. terraenovae.
In the dorsal protocerebrum, DNl in P. terraenovae
seems to correspond to DN3 in D. melanogaster. However,
only four to six DNls were stained, which are much fewer than the
DN3s (up to 40 cells) in D. melanogaster. In the
medio-dorsal protocerebrum of D. melanogaster, two groups,
DN1 and DN2, have been identified, with different fibre
projections (Kaneko and Hall,
2000). P. terraenovae may also have two clusters in
DNm, but discrimination of the two is difficult without
identification of fibre projections. Also fewer DN clusters have been reported
in M. domestica than in D. melanogaster
(Codd et al., 2007). Although
there are some differences from D. melanogaster, it seems that P.
terraenovae retains a homologous set of PER-ir neurons, as does M.
domestica. These neural networks might be common to all the dipteran
Cyclorrhapha.
In P. terraenovae, nuclear staining at ZT 0–1 suggests that
PER protein enters the nucleus and there is a possibility that PER plays some
role in regulation of clock gene expressions, as in D. melanogaster
(Curtin et al., 1995;
Shafer et al., 2004). Analysis
of other circadian clock genes is required in future studies.
s-LNvs are important for circadian activity rhythms
Molecular tools are not available for P. terraenovae; therefore,
the roles of PER-ir neurons in circadian activity rhythms and photoperiodism
were examined by microsurgery. We focused on LNvs as their
importance for activity rhythms under DD was shown in D. melanogaster
(Helfrich-Förster, 2003).
The incidence of rhythmic patterns and the number of small PDF somata
(s-LNvs) remaining after surgery correlated, whereas no
correlations were observed between the number of large PDF somata
(l-LNvs) and activity patterns
(Fig. 8B). The results suggest
that s-LNvs are required to drive locomotor activity rhythms under
DD. Only one fly showed rhythmicity when all small PDF somata were missing.
This might be caused by other per-expressing neurons. It is suggested
in D. melanogaster that PDF-negative lateral neurons (LNds and one
sLNv) or a subset of DNs control rhythmic behaviour under constant light
conditions (Rieger et al.,
2006; Murad et al.,
2007; Picot et al.,
2007). If it is also true in P. terraenovae then these
neurons might drive the activity rhythm even under DD if the cellular networks
were affected by the surgery.
Compared with DNs, the importance of LNs as pacemaker neurons for activity
rhythms has been shown in D. melanogaster
(Frisch et al., 1994;
Helfrich-Förster, 1998;
Grima et al., 2004;
Stoleru et al., 2004), and the
dominant roles of s-LNvs in driving circadian activity rhythms have
been reported (Stoleru et al.,
2005; Helfrich-Förster et
al., 2007). Our results in P. terraenovae support those
observations. Because LNds are located far dorsal to the
s-LNvs, it is unlikely that s-LNvs ablation invaded the
LNds regions. We suggest, therefore, that LNds are not
capable of driving activity rhythms without the presence of s-LNvs
under DD. Although the possibility that LNds plays a role in
activity rhythms cannot be excluded, we consider LNds to play a
less dominant role than s-LNvs under DD.
After removal of the anterior base of the medulla, residual rhythms were
observed for a few days in some flies (Fig.
5D). Similar patterns have been reported in behaviourally
arrhythmic disconnected mutant D. melanogaster in which
PDF-ir neurons are missing
(Helfrich-Förster and Homberg,
1993; Wheeler et al.,
1993; Helfrich-Förster,
1998). Residual rhythms in disconnected flies appeared
for several days under DD, although per0 mutant flies are
completely arrhythmic under DD (Wheeler et
al., 1993). Such residual rhythms could be explained by the
presence of other per-expressing neurons.
P. terraenovae exhibits diurnal locomotor activity rhythms
(Hamasaka et al., 2002). After
the site 2 operations, however, some flies were nocturnally active under LD
conditions, irrespective of their activity pattern under DD. In D.
melanogaster the importance of l-LNvs in light-arousal
activities by altering electrical activities of the LNv has been
reported just recently (Sheeba et al.,
2008; Shang et al.,
2008). D. melanogaster shows bimodal rhythms with morning
and evening activities. When LNvs were hyper-excited, enhancement
of the nocturnal locomotor activity was observed and the normal
day–night firing patterns of the action potentials in l-LNvs
were reversed (Sheeba et al.,
2008; Shang et al.,
2008). Various crosses of transgenic flies demonstrated that the
enhancement of nocturnal activities was due to hyper-excitement in
l-LNvs, and it occurred even without s-LNvs, which is necessary for
circadian oscillation in DD (Sheeba et
al., 2008; Shang et al.,
2008). l-LNvs are suggested to modulate arousal and
sleep in clock-independent manner, and excitement of l-LNv might be
inhibited during the dark period in D. melanogaster. In P.
terraenovae also l-LNvs may control the activity level: these
neurons are excited during the light period and inhibited during the dark
period. It may be that in flies in which the activity pattern showed the
masking effects after site 2 operation the surgery did not remove
l-LNvs but caused damage to the neural circuit inhibiting
l-LNvs during the dark period. Therefore, nocturnal high activities
could be observed.
A plausible involvement of s-LNvs in photoperiodism
Flies lacking s-LNvs (site 4 operation) did not discriminate
long days from short days, and diapause incidence was about 50% under both
photoperiodic conditions. Because the site 3 (control) operation did not
affect photoperiodism, the effects of the site 4 operation were not the result
of damage to any brain tissue. This suggests that the tissue of the site 4
contained neurons playing some roles in photoperiodism.
One may surmise that absence of neurons important for photoperiodic
induction of diapause will result in entire non-diapause or diapause
phenotypes. Actually, removal of pars lateralis neurons causes non-diapause
phenotypes even under diapause-inducing conditions in some insects, including
P. terraenovae (e.g. Shiga and
Numata, 2000; Shimokawa et
al., 2008) (for a review, see
Shiga and Numata, 2007). The
pars lateralis neurons innervating the corpus cardiacum or corpus allatum have
been considered to inhibit hormonal events necessary for reproduction or
development under diapause-inducing conditions. Among the three components of
photoperiodism (the photoreceptor, the photoperiodic clock, and the effecter),
the pars lateralis neurons might be located in the effecter control centre.
However, removal of a region containing s-LNvs in P.
terraenovae resulted in almost equal occurrence of diapause and
non-diapause phenotypes. When there are no output signals from the
photoperiodic clock to the effecter, because of a lack of photoperiodic clock
components, the diapause phenotype could occur at random and its incidence
would be expected to be about 50%. This was observed in our results and
suggests that the region containing s-LNvs is involved in the
photoperiodic clock.
Site 4 contained s-LNvs but no correlations were found between
diapause incidence and the number of s-LNvs removed. This
observation suggests two possible mechanisms: (1) neurons other than
s-LNvs in site 4 are important for the photoperiodic mechanism or
(2) s-LNvs neurons are involved in the photoperiodic mechanism.
There was a correlation between circadian rhythmicity and the number of s-LNv
(Fig. 8B). When many of the
s-LNvs remained, circadian rhythmicity remained but photoperiodism
disappeared, irrespective of the number of remaining s-LNvs. This
would support the first mechanism. Neural architecture, however, might be
different between circadian oscillation and photoperiodism. The photoperiodic
mechanism may require complex neuronal networks between s-LNvs or
between s-LNvs and other neurons in the proximity. Therefore, even
with substantial numbers of s-LNvs left, ablation of the region
proximal to the s-LNvs might cause disturbance of the photoperiodic
mechanism. In this scenario, the second mechanism is also probable. In a
previous study synaptic connections from s-LNvs to the pars lateralis neurons,
which is important for photoperiodic diapause, were demonstrated in P.
terraenovae (Hamanaka et al.,
2005). Thus, we consider that the s-LNvs might send
circadian timing information to the pars lateralis neurons to control the
diapause phenotype. Although the current study cannot determine which
possibility is more likely, it presents, for the first time, some evidence
that circadian clock neurons are involved in photoperiodism. Microsurgery
experiments are less accurate than molecular level techniques, however, such
surgery in combination with fine-scale neuroanatomy might resolve the neural
networks involved in the photoperiodic mechanism. The current results together
with previous studies by Shiga and Numata
(Shiga and Numata, 2000) and
Hamanaka et al. (Hamanaka et al.,
2005) suggest that a neural connection between s-LNvs
and pars lateralis neurons may be involved in photoperiodic mechanisms in
P. terraenovae.
In D. melanogaster, although critical day length was shifted to a
shorter value than in wild-type flies, adult females of the
per0 strain showed photoperiodic control of reproductive
diapause (Saunders et al.,
1989). Saunders et al.
(Saunders et al., 1989)
mentioned that the per locus is not causally involved in the time
measurement and that the crucial genes lie in different loci of the genome. In
C. costata, it has been shown that another clock gene
timeless is crucial for photoperiodic control of larval diapause. In
a non-photoperiodic-diapause (NPD) strain of C. costata, of which a
single autosomal gene locus encoding tim was mutated, both circadian
eclosion rhythms and photoperiodic control of larval diapause were lost
(Pavelka et al., 2003).
Analysis of tim mRNA and TIM protein in the larval brain indicated
that regulated transcription of tim in two brain neurons was required
for photoperiodic induction of diapause in C. costata
(Stehlik et al., 2008).
Studies on D. melanogaster have suggested that tim directly
affects the incidence of diapause through circadian photoreception
(Tauber et al., 2007;
Sandrelli et al., 2007). These
studies show involvement of circadian clock genes per and
tim in photoperiodic control of diapause. The present study examined
involvement of circadian clock neurons, and raised a possibility that
circadian clock neurons, s-LNvs, active in behavioural rhythms are
also involved in photoperiodism. Our data support an idea that circadian
behavioural rhythms and photoperiodism share neural elements in their
underlying mechanisms. An examination of molecular level events in
LNvs by comparing the expression patterns of per and
tim would be an interesting next step in resolving the relationships
between circadian rhythms and photoperiodism.
LIST OF ABBREVIATIONS
|
Footnotes |
|---|
|
References |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Baylies, M. K., Bargiello, T. A., Jackson, F. R. and Young, M. W. (1987). Changes in abundance or structure of the per gene product can alter periodicity of the Drosophila clock. Nature 326,390 -392.[CrossRef][Medline]
Bowen, M. F., Saunders, D. S., Bollenbacher, W. E. and Gilbert,
L. I. (1984). In vitro reprogramming of the
photoperiodic clock in an insect brain-retrocerebral complex. Proc.
Natl. Acad. Sci. USA 81,5881
-5884.
Codd, V., Dole
el, D., Stehlik, J., Piccin, A., Garner,
K. J., Racey, S. N., Straatman, K. R., Louis, E. J., Costa, R., Sauman, I. et
al. (2007). Circadian rhythm gene regulation in the housefly
Musca domestica. Genetics
177,1539
-1551.
Curtin, K. D., Huang, Z. J. and Rosbash, M. (1995). Temporally regulated nuclear entry of the Drosophila period protein contributes to the circadian clock. Neuron 14,365 -372.[CrossRef][Medline]
Frisch, B., Hardin, P. E., Hamblen-Coyle, M. J., Rosbash, M. and Hall, J. C. (1994). A promotorless period gene mediates behavioral rhythmicity and cyclical per expression in a restricted subset of the Drosophila nervous system. Neuron 12,553 -570.
Frisch, B., Fleissner, G., Fleissner, G., Brandes, C. and Hall, J. C. (1996). Staining in the brain of Pachymorpha sexguttata mediated by an antibody against a Drosophila clock-gene product: labeling of cells with possible importance for the beetle's circadian rhythms. Cell Tissue Res. 286,411 -429.[CrossRef][Medline]
Goto, S. G. and Denlinger, D. L. (2002). Short-day and long-day expression patterns of genes involved in the flesh fly clock mechanism: period, timeless, cycle and cryptochrome. J. Insect Physiol. 48,803 -816.[CrossRef][Medline]
Goto, S. and Numata, H. (2003). Expression patterns of genes involved in the blow fly clock mechanism. Zool. Sci. 20,1592 .
Grima, B., Chélot, E., Xia, R. and Rouyer, F. (2004). Morning and evening peaks of activity rely on different clock neurons of the Drosophila brain. Nature 431,869 -873.[CrossRef][Medline]
Hamanaka, Y., Yasuyama, K., Numata, H. and Shiga, S. (2005). Synaptic connections between pigment-dispersing factor-immunoreactive neurons and neurons in the pars lateralis of the blow fly, Protophormia terraenovae J. Comp. Neurol. 491,390 -399.[CrossRef][Medline]
Hamanaka, Y., Tanaka, S., Numata, H. and Shiga, S. (2007). Peptide immunocytochemistry of neurons projecting to the retrocerebral complex in the blow fly, Protophormia terraenovae.Cell Tissue Res. 329,581 -593.[CrossRef][Medline]
Hamasaka, Y., Watari, Y., Arai, T., Numata, H. and Shiga, S. (2002). Retinal and extraretinal pathways for entrainment of the circadian activity rhythm in the blow fly, Protophormia terraenovae.J. Insect Physiol. 47,867 -875.
Helfrich-Förster, C. (1995). The period
gene is expressed in CNS neurons which also produce a neuropeptide that
reveals the projections of circadian pacemaker cells within the brain of
Drosophila melanogaster. Proc. Natl. Acad. Sci. USA
92,612
-616.
Helfrich-Förster, C. (1998). Robust circadian rhythmicity of Drosophila melanogaster requires the presence of lateral neurons: a brain-behavioral study of disconnected mutants. J. Comp. Physiol. A 182,435 -453.[CrossRef][Medline]
Helfrich-Förster, C. (2003). The neuroarchtecture of the circadian clock in the brain of Drosophila melanogaster. Microsc. Res. Tech. 62, 94-102.[CrossRef][Medline]
Helfrich-Förster, C. and Homberg, U. (1993). Pigment-dispersing hormone-immunoreactive neurons in the nervous system of wild-type Drosophila melanogaster and of several mutants with altered circadian rhythmicity. J. Comp. Neurol. 337,177 -190.[CrossRef][Medline]
Helfrich-Förster, C., Yoshii, T., Wülbeck, C.,
Grieshaber, E., Rieger, D., Bachleitner, W., Cusamano, P. and Rouyer, F.
(2007). The lateral and dorsal neurons of Drosophila
melanogaster: new insights about their morphology and function.
Cold Spring Harb. Symp. Quant. Biol.
72,517
-525.
Hodková, M., Syrová, Z., Doleel, D. and
auman, I. (2003). Period gene expression in
relation to seasonality and circadian rhythms in the linden bug,
Pyrrhocoris apterus (Heteroptera). Eur. J.
Entomol. 100,267
-273.
Honda, T., Matsushima, A., Sumida, K., Chuman, Y., Sakaguchi, K., Onoue, H., Meinertzhagen, I. A., Shimohigashi, Y. and Shimohigashi, M. (2006). Structural isoforms of the circadian neuropeptide PDF expressed in the optic lobes of the cricket Gryllus bimaculatus: Immunocytochemical evidence from specific monoclonal antibodies. J. Comp. Neurol. 499,404 -421.[CrossRef][Medline]
Iwai, S., Fukui, Y., Fujiwara, Y. and Takeda, M. (2006). Structure and expression of two circadian clock genes, period and timeless in the commercial silk moth, Bombyx mori. J. Insect Physiol. 52,625 -637.[CrossRef][Medline]
Kaneko, M. and Hall, J. C. (2000). Neuroanatomy of cells expressing clock genes in Drosophila: transgenic manipulation of the period and timeless genes to mark the perikarya of circadian pacemaker neurons and their projections. J. Comp. Neurol. 422,66 -94.[CrossRef][Medline]
Konopka, R. J. and Benzer, S. (1971). Clock
mutants of Drosophila melanogaster. Proc. Nat. Acad. Sci.
USA 68,2112
-2116.
Matsuo, J., Nakayama, S. and Numata, H. (1997). Role of the corpus allatum in the control of adult diapause in the blow fly, Protophormia terraenovae. J. Insect Physiol. 43,211 -216.[CrossRef][Medline]
Moriyama, Y., Sakamoto, T., Karpova, S. G., Matsumoto, A., Noji,
S. and Tomioka, K. (2008). RNA interference of the clock gene
period disrupts circadian rhythms in the cricket Gryllus
bimaculatus. J. Biol. Rhythms
23,308
-318.
Murad, A., Emery-Le, M. and Emery, P. (2007). A subset of dorsal neurons modulates circadian behavior and light responses in Drosophila. Neuron 53,689 -701.[CrossRef][Medline]
Nakao, N., Ono, H., Yamamura, T., Anraku, T., Takagi, T., Higashi, K., Yasuo, S., Katou, Y., Kageyama, S., Uno, Y. et al. (2008). Thyrotrophin in the pars tuberalis triggers photoperiodic response. Nature 452,317 -323.[CrossRef][Medline]
Nässel, D. R., Shiga, S., Mohrherr, C. J. and Rao, K. R. (1993). Pigment-dispersing hormone-like peptide in the nervous system of the flies Phormia and Drosophila: immunocytochemistry and partial characterization. J. Comp. Neurol. 331,183 -198.[CrossRef][Medline]
Numata, H. and Shiga, S. (1995). Induction of adult diapause by photoperiod and temperature in Protophormia terraenovae (Diptera: Calliphoridae) in Japan. Environ. Entomol. 24,1633 -1636.
Pavelka, J., Shimada, K. and Kostal, V. (2003). Timeless: a link between fly's circadian and photoperiodic clocks? Eur. J. Entomol. 100,255 -265.
Picot, M., Cusumano, P., Klarsfeld, A., Ueda, R. and Rouyer, F. (2007). Light activates output from evening neurons and inhibits output from morning neurons in the Drosophila circadian clock. PLOS Biol. 5,2513 -2521.
Plautz, J. D., Kaneko, M., Hall, J. C. and Kay, S. A.
(1997). Independent photoreceptive circadian clocks throughout
Drosophila. Science 278,1632
-1635.
Rieger, D., Shafer, O. T., Tomioka, K. and
Helfrich-Förster, C. (2006). Functional analysis of
circadian pacemaker neurons in Drosophila melanogaster. J.
Neurosci. 26,2531
-2543.
Riihimaa, A. J. and Kimura, M. T. (1989). Genetics of the photoperiodic larval diapause in Chymomyza costata (Diptera: Drosophilidae). Hereditas 110,193 -200.[CrossRef]
Sandrelli, F., Tauber, E., Pegoraro, M., Mozzotta, G., Cisotto,
P., Landskron, J., Stanewsky, R., Piccin, A., Rosato, E., Zordan, M. et
al. (2007). A molecular basis for natural selection at the
timeless locus in Drosophila melanogaster.Science 316,1898
-1900.
Sauman, I. and Reppert, S. M. (1996). Circadian clock neurons in the silkmoth Antheraea pernyi: novel mechanisms of period protein regulation. Neuron 17,889 -900.[CrossRef][Medline]
Saunders, D. S. (2002). Insect Clocks. 3rd edn. Amsterdam: Elsevier.
Saunders, D. S., Henrich, V. C. and Gilbert, L. I.
(1989). Induction of diapause in Drosophila
melanogaster: photoperiodic regulation and the impact of arrhythmic clock
mutations on time measurement. Proc. Natl. Acad. Sci.
USA 86,3748
-3752.
Shafer, O. T., Levine, J. D., Truman, J. W. and Hall, J. C. (2004). Flies by night: effects of changing day length on Drosophila's circadian clock. Curr. Biol. 14,424 -432.[Medline]
Shang, Y., Griffith, L. C. and Rosbash, M.
(2008). Light-arousal and circadian photoreception circuits
intersect at the large PDF cells of the Drosophila brain. Proc.
Natl. Acad. Sci. USA 105,19587
-19594.
Sheeba, V., Fogle, K. J., Kaneko, M., Rashid, S., Chou, Y.-T., Sharma, V. K. and Holmes, T. C. (2008). Large ventral lateral neurons modulate arousal and sleep in Drosophila. Curr. Biol. 18,1537 -1545.[CrossRef][Medline]
Shiga, S. and Numata, H. (2000). The roles of neurosecretory neurons in the pars intercerebralis and pars lateralis in reproductive diapause of the blow fly, Protophormia terraenovae.Naturwissenschaften 87,125 -128.[CrossRef][Medline]
Shiga, S. and Numata, H. (2007). Neuroanatomical approaches for insect photoperiodism. Photochem. Photobiol. 83,76 -86.[Medline]
Shiga. S., Davis, N. T. and Hildebrand, J. G. (2003). Role of neurosecretory cells in the photoperiodic induction of pupal diapause of the tobacco hornworm Manduca sexta.J. Comp. Neurol. 462,275 -285.[CrossRef][Medline]
Shimokawa, K., Numata, H. and Shiga, S. (2008). Neurons important for the photoperiodic control of diapauses in the bean bug, Riptortus pedestris J. Comp. Physiol. A 194,751 -762.[CrossRef][Medline]
Sokolove, P. G. and Bushell, W. N. (1978). The chi square periodogram: its utility for analysis of circadian rhythms. J. Theor. Biol. 72,131 -160.[CrossRef][Medline]
Stanewsky, R. (2003). Genetic analysis of the circadian system in Drosophila melanogaster and mammals. J. Neurobiol. 54,111 -147.[CrossRef][Medline]
Stehlik, J., Závodská, R., Shimada, K.,
auman, I. and Ko
tál, V. (2008).
Photoperiodic induction of diapause requires regulated transcription of
timeless in the larval brain of Chymomyza costata. J.
Biol. Rhythms 23,129
-139.
Stoleru, D., Peng, Y., Agosto, J. and Rosbash, M. (2004). Coupled oscillators control morning and evening locomotor behaviour of Drosophila. Nature 431,862 -868.[CrossRef][Medline]
Stoleru, D., Peng, Y., Nawathean, P. and Rosbash, M. (2005). A resetting signal between Drosophila pacemakers synchronizes morning and evening activity. Nature 438,238 -242.[CrossRef][Medline]
Tauber, E., Zordan, M., Sandrelli, F., Pegoraro, M.,
Osterwalder, N., Breda, C., Daga, A., Selmin, A., Monger, K., Benna, C. et
al. (2007). Natural selection favors a newly derived
timeless allele in Drosophila melanogaster.Science 316,1895
-1898.
Turck, F., Fornara, F. and Coupland, G. (2008). Regulation and identity of florigen FLOWERING LOCUS T moves center stage. Annu. Rev. Plant Biol. 59,573 -594.[CrossRef][Medline]
Vaz Nunes, M. and Saunders, D. (1999).
Photoperiodic time measurement in insects: a review of clock models.
J. Biol. Rhythms 14,84
-104.
Veerman, A. (2001). Photoperiodic time measurement in insects and mites: a critical evaluation of the oscillator-clock hypothesis. J. Insect Physiol. 47,1097 -1109.[CrossRef][Medline]
Wheeler, D. A., Hamblen-Coyle, M. J., Dushay, M. S. and Hall, J.
C. (1993). Behavioral in light-dark cycles of
Drosophila mutants that are arrhythmic, blind, or both. J.
Biol. Rhythms 8,67
-94.
Wise, S., Davis, N. T., Tyndale, E., Noveral, J., Folwell, M. G., Bedian, V., Emery, I. F. and Siwicki, K. (2002). Neuroanatomical studies of period gene expressio in the hawkmoth, Manduca sexta. J. Comp. Neurol. 447,366 -380.[CrossRef][Medline]
Yasuo, S., Watanabe, M., iigo, M., Yamamura, T., Nakao, N., Takagi, T., Ebihara, S. and Yoshimura, T. (2006). Molecular mechanism of photoperiodic time measurement in the brain of Japanese quail. Chronobiol. Int. 23,307 -315.[CrossRef][Medline]
Zar, J. H. (1999). Biostatistical Analysis, 4th edn. Upper Saddle River, NJ: Prentice Hall.
Závodská, R., Sehadová, H., Sauman, I. and Sehnal, F. (2005). Light-dependent PER-like proteins in the cephalic ganglia of an apterygote and a pterygote insect species. Histochem. Cell Biol. 123,407 -418.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in JEB:
This article has been cited by other articles:
|
|
 |
|
  K. Knight CIRCADIAN AND PHOTOPERIOD CLOCKS SHARE NEURONS J. Exp. Biol., March 15, 2009; 212(6): iii - iii. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
