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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
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Roles of PER immunoreactive neurons in circadian rhythms and photoperiodism in the blow fly, Protophormia terraenovae

Sakiko Shiga* and Hideharu Numata

Department of Biology and Geosciences, Graduate School of Science, Osaka City University, Sumiyoshi, Osaka 558-8585, Japan


Figure 1
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  		Fig. 1. Schematic illustrations of ablated regions in the adult brain of Protophormia terraenovae. Four large pigment-dispersing factor (PDF)-immunoreactive somata (large filled circles), four small PDF-immunoreactive somata (small filled circles), and their fibres are shown bilaterally in dorsal (upper) and frontal (lower) views. Ablated regions are encircled with dotted lines. (A) For examination of activity rhythms, site 1 (control) and site 2 (test) were ablated (site 1 and site 2 operations, respectively). Note that site 1 is located anterior to the PDF-immunoreactive fibres and does not include those fibres. (B) For examination of photoperiodism, site 3 (control) and site 4 (test) were ablated (site 3 and site 4 operations, respectively). Br, brain; E, oesophageal foramen; OL, optic lobe; SEG, suboesophageal ganglion.

 

Figure 2
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  		Fig. 2. Photomicrographs of PER-immunoreactive cells in the adult brain of Protophormia terraenovae (whole-mounts, frontal views, dorsal region to the top and medial to the right). (A) In an anterior ventral boundary between the left optic lobe and mid-brain, a cluster of four large ventral lateral neurons (l-LNvs) and five small ventral lateral neurons (s-LNvs) are shown. A2 is an enlarged image of the boxed region in A1. Arrows indicate five s-LNvs. (B) In an anterior dorsal boundary between the left optic lobe and mid-brain, four dorsal lateral neurons (LNds) are in focus. (C) The dorsal posterior region of the protocerebrum, showing a stained cluster of lateral dorsal neurons (DNls) and a stained cluster of medial dorsal neurons (DNms). Only part of DNls and DNms are in focus. Scale bars, 100 µm.

 

Figure 3
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  		Fig. 3. Tracings of PER-immunoreactive cells in a left hemisphere in two representative adult brains of Protophormia terraenovae. See legend of Fig. 2 for abbreviations. Open and filled circles indicate cells located in anterior and posterior regions, respectively. Scale bar, 100 µm.

 

Figure 4
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  		Fig. 4. Photomicrographs of neurons labelled by PER and pigment-dispersing factor (PDF) antisera in the adult brain of Protophormia terraenovae. Stained neurons were viewed as bright-field images for PER-immunoreactive cells (A1, B1 and C1) and the exactly same areas at the same depth was examined as fluorescent images for PDF-immunoreactive neurons (A2, B2 and C2). Note that the cytoplasm and fibres were labelled using the PDF antiserum, whereas the PER antiserum labelled only somata. Arrowheads are located in exactly the same positions in the respective right and left panels. (A) Four s-LNvs (single arrowheads). (B) Two l-LNvs (double arrowheads) and two s-LNvs (single arrowhead). Note that one s-LNv is not labelled by the PDF antiserum. (C) Four DNms (single arrowheads) are present, but only one DNm is labelled by the PDF antiserum. Scale bar, 50 µm.

 

Figure 5
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  		Fig. 5. Locomotor activity rhythms in flies after sham operation (A), site 1 operation (B) and site 2 operation (C,D) in adult Protophormia terraenovae. Locomotor activities were recorded under constant darkness (DD) for 7–8 days and under 12 h:12 h L:D for 9 days. Activities are shown in double plotted actograms with associated periodogram analysis results. White and black bars at the bottom of actograms indicate respective light and dark periods. Flies after sham or site 1 operations showed rhythmic patterns (A,B), but arrhythmic (C) and obscure patterns (D) appeared after site 2 operations.

 

Figure 6
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  		Fig. 6. Diel activity levels in intact, sham-operated, site 1-operated and site 2-operated adult Protophormia terraenovae under 12 h:12 h and 18 h:6 h L:D. Relative activity levels were generated as described in the text. Each column corresponds to a 30-min period. The open columns represent relative activity levels during the photophase, and the filled columns those during the scotophase. Bars above each column show s.e.m. (i.e. fly-to-fly variation in relative activity level for a given period).

 

Figure 7
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  		Fig. 7. Representative tracings of PDF-immunoreactive neurons in the adult brains of Protophormia terraenovae. Top left is an example of the intact group and the others are five of the site 2-operated group. In the intact brain, four small (black arrows) and four large PDF somata (white arrows) were bilaterally stained. In the site 2-operated examples different numbers of PDF somata remained. Scale bar, 100 µm.

 

Figure 8
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  		Fig. 8. Effects of the surgical operation on rhythmicity under constant darkness in adult Protophormia terraenovae. (A) In intact, sham-operated and site 1-operated (control) groups, most females showed rhythmic patterns. After a site 2 operation, however, locomotor activities were arrhythmic in 25.8% and obscure in 29% of flies. Different letters above the columns indicate significant differences in incidences of females with rhythmic patterns (Tukey-type multiple comparison test for proportions, P<0.05). (B) Results after site 2 operations are shown according to the number of remaining PDF somata. The number of PDF somata was counted in each fly, and flies were grouped according to the number of large PDF somata (left) and small PDF somata (right). There were no flies in which all large (N=8) or all small PDF somata (N=8) remained. Incidences of the rhythmic patterns depended on the number of remaining small PDF somata; the fewer small PDF somata remaining, the fewer flies that showed rhythmic patterns.

 

Figure 9
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  		Fig. 9. Effects of the surgical operation on photoperiodism in adult Protophormia terraenovae. (A) In intact, sham-operated, and site 3-operated groups, most females entered diapause under 12 h:12 h L:D, whereas most females developed ovaries under 18 h:6 h L:D. After site 4 operation, flies did not show photoperiodism, and diapause incidences were 55.1% under 12 h:12 h L:D and 48.4% under18 h:6 h L:D. (B) Results after site 4 operation are shown according to the number of remaining small PDF somata. Filled and open columns show reproductive females and females in diapause, respectively. Different letters above the columns indicate significant differences in incidences of females in diapause (Tukey-type multiple comparison test for proportions, P<0.05).

 

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