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INSECT TIMING:
CIRCADIAN
RHYTHMICITY TO
SEASONALITY

This Page Intentionally Left Blank

TIMING:
CIRCADIAN
RHYTHMICITY TO
SEASONALITY
Edited by:
D.L. Denlinger
J.M. Giebultowicz
D.S. Saunders
2001
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Preface
The time of day and season of the year are among the most profound environmental
factors dictating daily and seasonal patterns of insect activity. We hope that the analysis of
insect timing presented in this book will prove helpful in portraying recent developments in
this exciting field of research. We are grateful to the organizers of the XXI International
Congress of Entomology, held in Iguassu Falls, Brazil, August 20-26, 2000, for hosting
three interrelated symposia that formed the basis for this publication. The three symposia
included "Circadian Clocks in Insects: Molecular and Cellular Perspectives" organized by
J. M. Giebultowicz and M. D. Marques, "Photoperiodic Induction of Diapause and
Seasonal Morphs" organized by D. S. Saunders and H. Numata, and "New Complexities
in the Regulation of Insect Diapause and Cold Hardiness" organized by D. L. Denlinger
and O. Yamashita. Chapters in the book thus range from discussions on the molecular
regulation of circadian rhythms to physiological events associated with successful
overwintering strategies. We appreciate the enthusiasm of the authors for this project and
their timely submission of manuscripts.
We are grateful to Sandra Migchielsen and Ken Plaxton from Elsevier for their
encouragement and advice, and to Irene Lenhart for her assistance in compiling the index.
D.L. Denlinger
J.M. Giebultowicz
D.S. Saunders

Foreword
James W. Truman
Department of Zoology, University of Washington, Box 351800, Seattle, WA 98195
USA
Insects, like other organisms, have evolved a diverse array of physiological and
behavioral mechanisms to allow them to cope with both biotic and abiotic challenges.
These mechanisms, though, are not used simply in a reactive mode that only rises to meet
challenges once they are presented. An intriguing aspect of life is that organisms
continually make predictions about the future and adjust their physiology and behavior to
anticipate the changes that will occur in the world around them. One class of predictive
mechanisms used by insects to deal with their complex biological world involves learning
processes that allow cause and effect associations to be made based on experience. To
make predictions about their abiotic world, insects use mechanisms that take advantage of
predictable aspects of the physical world, notably the geophysical rhythms of the daily
rotation of the earth about its axis, the orbiting of the moon around the earth, and the
seasonal journey of the earth around the sun. These timekeeping systems are not essential
for life per se, as long as life occurs in the predictable environment of the laboratory. In the
natural world, though, these timing systems fine-tune the responses of the animal to
optimize its performance since physiological or behavioral mechanisms can gear-up in
anticipation of expected changes in the world, rather than always being in a reactive mode.
The mechanisms that insects have used to deal with the daily and seasonalfluctuationsof
their world have fascinated physiologists and behaviorists through the latter half of the
20th century. Research by pioneers, such as C.S. Pittendrigh, C.M.Williams, A.D. Lees,
S. Beck, J. deWilde, and A.S. Danilevskii, has illustrated the diversity and richness of
phenomena relating to the adaptation of insects to daily and seasonal cycles. These
researchers also showed that the study of these processes could produce fundamental
insights into diverse areas of insect physiology, ecology and behavior.
This book brings together three topics that are central to the understanding of biological
timing in insects: circadian rhythms, photoperiodism and diapause. The study of circadian
rhythms has undergone a flowering in recent years with the molecular dissection of the
components of the circadian clock. Research on diverse systems ranging from plants and
fungi to insects and vertebrates shows that the molecular strategy for constructing a
molecular clock is quite conserved as are some of the molecules that compose the clock.
The molecular dissection of circadian clocks has open up a number of exciting new areas.
One is aimed at a deeper understanding of the manner by which entraining signals interact
with the molecular oscillation that comprises the workings of the clock. In addition, the
issue of how the molecular oscillation is coupled with cellular outputs is a fascinating
question that is just beginning to be addressed. A second area involves a comparative
approach in which the identification of clock-related genes in Drosophila has provided the
molecular tools to bootstrap into the clock molecules of a diversity of insects. On the
physiological side, perhaps the most exciting results come using the localization of clock
proteins and their messenger RNAs to establish the cellular locations of putative clocks. It

Vlll
is now clear that one can no longer talk of a "master clock" that is responsible for the
complete temporal organization of the animal. Organisms are more like "clock shops" with
numerous cellular oscillators. In this context, though, not all clocks are created equal and
we need to understand the nature and logic of the timing hierarchies. Since selected insect
tissues can retain their rhythmical activities under organ culture conditions, insects are
providing premier systems for studying how the hierarchical organization of clocks results
in the overall temporal organization of the animal. The area of insect photoperiodism has
not yet reached the level of molecular understanding that is seen for circadian rhythms.
Nevertheless, photoperiodic phenomena continue to provide a fertile ground for the
physiologist, ecologist and evolutionary biologist. Perhaps more than any trait,
photoperiodism represents the "stamp" of the planet on a population of organisms.
In insects, the most obvious manifestation of photoperiodism is diapause. Here too,
there is a theme of both unity and diversity. The spectrum of diapausing stages in insects,
ranging from the Qgg to the adult opens up a spectrum of endocrine controls, a few of
which are still being resolved. The focus of the field, though, is towards the cellular and
molecular changes that occur as an insect makes the transition into and out of diapause. •
Overall, the volume presents the rich diversity of challenges and opportunities provided
by insects for the study of how life has adapted to the rhythms of the planet. It provides a
good platform for the movement of the field into the new century.

TABLE OF CONTENTS
Page
Preface v
Foreword
J. W. Truman vii
1. The blow fly Calliphora vicina: a "clock-work" insect
D. S. Saunders 1
2. Molecular control of Drosophila circadian rhythms
P. Schotland and A. Sehgal 15
3. Organization of the insect circadian system: spatial and
developmental expression of clock genes in peripheral tissues
of Drosophila melcmogaster
J. M. Giebultowicz, M. Ivanchenko and T. Vollintine 31
4. The circadian clock system of hemimetabolous insects
K. Tomioka, A. S. M. Saifullah and M. Koga 43
5. Cellular circadian rhythms in the fly's visual system
E. Pyza 55
6. Anatomy and functions of the brain neurosecretory neurons
with regard to reproductive diapause in the blow fly
Protopkormia terraenovae
S. Shiga and H. Numata 69
7. Photoperiodism and seasonality in aphids
J. Hardie 85
8. Photoperiodic time measurement and shift of the critical
photoperiod for diapause induction in a moth
S. Masaki and Y. Kimura 95
9. Geographical strains and selection for the diapause trait in Calliphora vicina
D. S. Saunders 113
10. Evolutionary aspects of photoperiodism in Drosophila
M. T. Kimura 123
11. Molecular analysis of overwintering diapause
S. R. Palli, R. Kothapalli, Q, Feng, T. Ladd, S. C. Perera,
S.-C. Zheng, K. Gojtan, A. S. D. Pang, M. Primavera,
W. Tomkins and A. Retnakaran 133

Insights for future studies on embryonic diapause promoted by
molecular analyses of diapause hormone and its action in Bombyx mori
O. Yamashita, K. Shiomi, Y. Ishida, N. Katagiri and T. Niimi 145
13. Stress proteins: a role in insect diapause?
D. L. Denlinger, J. P. Rinehart and G. D. Yocum 155
14. Regulation of the cell cycle during diapause
S. P. Tammariello 173
15. Significance of specific factors produced throughout diapause
in pharate first instar larvae and adults
K. Suzuki, H. Tanaka and Y. An 185
16. Surviving winter with antifreeze proteins: studies on budworms and beetles
V. K. Walker, M. J. Kuiper, M. G. Tyshenko, D. Doucet,
S. P. Graether, Y. -C. Liou, B. D. Sykes, Z. Jia, P. L. Davies
and L. A. Graham 199
17. Using ice-nucleating bacteria to reduce winter survival of Colorado
potato beetles: development of a novel strategy for biological control
R. E. Lee, Jr., L. A. Castrillo, M. R. Lee, J. A. Wyman
and J. P. Costanzo 213
Species index 229
Subject index 231

Insect Timing: Circadian Rhythmicity to Seasonality
D.L. Denlinger, J. Giebultowicz and D.S. Saunders (Editors)
© 2001 Elsevier Science B.V. All rights reserved.
The blow fly Calliphora vicina: a "clock-work" insect
D. S. Saunders
Institute of Cell, Animal and Population Biology, University of Edinburgh, West Mains Road,
Edinburgh EH9 3JT, Scotland, United Kingdom
The blow fly Calliphora vicina is used to illustrate the basic properties of the circadian
system and photoperiodic induction of seasonality. In the adult female fly, a daily rhythm of
locomotor activity and the (maternal) photoperiodic induction of larval diapause operate
simultaneously but show features suggesting that, although both are circadian based, they are
regulated by separate 'clocks' with dissimilar properties. The locomotor activity rhythm free-
runs in darkness with a persistent, temperature compensated period (x ~ 22.5 hours); the
circadian oscillations underlying photoperiodic time measurement, however, show a period
closer to 24 hours and evidence of rapid damping. Although both clocks are brain-centred and
may be entrained by extra-optic photoreceptors, they are probably located in separate sets of
neurons. Some problems in circadian and photoperiodic biology are enumerated.
1. INTRODUCTION
When surveying the remarkable clock-like properties of circadian rhythms it becomes
understandable why early biologists found them, at best, extraordinary. However, after more
than 50 years of intensive investigation - together with a modem knowledge of genetics and
evolutionary biology - we now know that endogenous circadian rhythms are the products of
natural selection. Circadian rhythms were probably 'invented' by early photosynthetic
procaryotes, and evolved in the face of the pervasive environmental fluctuations of light and
temperature associated with the rotation of the earth around its axis. They may now be seen in
organisms from cyanobacteria to 'higher' vertebrates; in eucaryotes they are probably almost
universal. Other endogenous rhythms have also evolved: for example, in inter-tidal organisms,
or in those exposed to the sometimes extreme annual fluctuations in day length, temperature
and humidity associated with the earth's rotation around the sun. Seasonal photoperiodism, one
such case, is probably one of the most important ftmctions of the circadian system in higher
organisms.
The circadian system presents a number of defining properties. These are: (1) 'daily'
rhythms that persist ('free-run') in the absence of solar day signals (thereby attesting to their
endogeneity). (2) a free-nmning rhythm (period x hours) which is close to but rarely equal to
24 hours. (3) a well-developed homeostasis of x when exposed to a wide range of physical,
chemical and biological variables, most notably temperature (i.e. they are temperature-
compensated). And (4) an ability to synchronise (entrain) to solar day variables, particularly
light and temperature, so that x becomes equal to the period of the entraining agent (or
Zeitgeber), and adopts an adaptive phase relationship to it. Photoperiodism, itself based on the
circadian system, also shows - in a covert manner - these defining properties. It provides the
organism with a 'clock' which discriminates between the long days (or short nights) of summer

and the short days (or long nights) of autumn and winter, thereby obtaining information on
calendar time from the environment. A wide range of organisms, principally higher plants and
animals living in terrestrial habitats at higher latitudes, use such 'noise-free' information to
control seasonally appropriate switches in metabolism (e.g. flowering, diapause, breeding,
migration), most of which have a clear functional significance or survival value.
The purpose of this chapter is to present these defining properties in an insect that
presents robust circadian rhythms (of locomotor activity) and photoperiodic regulation (of
diapause), with both clocks operating simultaneously. The insect of choice is the blow fly or
bluebottle, Calliphora vicina. It is intended that this chapter should act as an introduction to the
papers in this volume addressing current problems in circadian rhythms, photoperiodism,
diapause and insect seasonality.
2. LOCOMOTOR RHYTHMICITY
2.1. Free-running locomotor rhythmicity in continuous conditions
Adult blow flies show typically robust rhythms of locomotor activity in continuous dark-
ness (DD). The record shown in Fig. 1, and others like it, was obtained by placing a single
female fly in a 9 cm Petri dish with a supply of sugar and water. The dish was then positioned
in a vertical infra-red light beam in such a way that the moving fly interrupted the light.
Interruptions of the light beam were recorded by computer and later assembled into the
familiar 'double-plotted' actogram. The records obtained are typical for circadian activity
rhythms in individual insects (Saimders, 1982) and will be described only briefly in this paper.
48 h
Figure 1. Locomotor activity rhythm of an adult blow fly (Calliphora vicina) in darkness (DD)
at 20°C (double plotted actogram), showing afree-runningrhythm (T = 22.63 h).
In continuous darkness at 20°G, flies showed a persistent, 'noise-free' and 'free-running'
rhythm of activity and rest, with a period (i) generally less than 24 hours. Among groups of
several hundred flies, the mean value of T was found to be about 22.5 h (Cymborowski et al.,
1993; Hong and Saunders, 1998), although the range was from little over 21 h, to the longest
value, just over 25 h (Fig. 2). Persistence of this rhythm for the life time of the fly (up to 7
weeks) and, of course, departure of T from exactly 24 hours, provided cogent evidence for the
endogeneity of the rhythmic mechanism underlying such activity.

2 5
T 22-53
I
X
m
n r A -O-
21 22 23 24 25
Circadian period (T), h
Figure 2. Distribution of free-running periods (T values) for the locomotor activity rhythm of
aduh Calliphora vicina in DD at 20°C. Mean value of T = 22.53 0.78 h (N = 78).
Rhythms of locomotor activity also continue under continuous illumination (LL), at least
at low levels of irradiance. At low intensity (up to about 0.018 Wm""^) rhythmicity persisted but
T lengthened systematically, up to about 26 hours. Above about 0.033 Wm"^ (Fig. 3) flies
Day
Timeh
15.00 3.00 15.00 3.00 15.00
1 i
7UkiUUL T)A.iiinii.:iLi.,ii...
liiLiiJoi. . i L a i
iJii.U Jte,l.
idM l..l<.iiî.iJ >iM.. . :
: . 'ÂH.LH^MI v^.h..
iUlUi III iMt.JLm,.
14:,,. •• l,iltU»»>MitUi»
ay4iii,iîmi>iilrditii'in.i ii ; »iii,iiiii'»
^ Mil '.fill AtLîmtl 111 till-
28
i
_iDiL
JLllL
JM.
iSLi
I Ii
. iM .
JuiJilL
_iillU_
_Jlilli-
Figure 3. Locomotor activity rhythm of C vicina: (a) DD, (b) behavioural arrhythmicity under
LL at 0.33 Wm"^ (c) DD, all at 20°C. Arrows show times of transfer from DD to LL and vice
versa.

became behaviourally arrhythmic, although a free-nmning rhythm reappeared after the light
was extinguished (Hong and Saunders, 1994). Under an intermediate irradiance (about 0.024
Wm"'^) flies were initially arrhythmic, but then assumed a long-period rhythm, perhaps after
light adaptation of the photoreceptors.
The majority (82.9 per cent) of flies under continuous darkness showed simple (unimodal)
free-running rhythms of activity; the remainder showed more complex patterns. In some (7.3
per cent), spontaneous and abrupt changes in x occurred, either to a longer or to a shorter value.
About 5 per cent were behaviourally arrhythmic, and a fiirther 5 per cent showed complex
patterns with either bimodality or internal desynchronisation into two or more 'bands' of
activity, free-running with different values of x (Kenny and Saunders, 1991; Hong and
Saunders, 1998). The significance of these complex patterns, and others, will be considered
below (Section 4).
2.2. Temperature compensation of the circadian period
At constant temperatures between 15 and 25°C the free-running period in DD (XDD) was
temperature compensated, showing Qio values between 0.98 and 1.04 (Saunders and Hong,
2000). Single temperature steps-up (from 20 to 25°C) or steps-down (from 20 to 15°C),
however, caused stable phase shifts of the activity rhythm (Fig. 4). Phase advances were
dominant for steps-up and phase delays for steps-down. Phase response curves for the two
types of signal were almost 'mirror images' of each other. Entrainment by temperature (and
light) will be considered below.
Timeh
14.00 02.00 14.00 02.00 14.00
Day ^ 4_u^
Figure 4. Locomotor activity rhythm of a female C vicina under (a) 20°C, (b) 15°C and (c)
20°C, all in DD, showing phase shifts but no change in period.

2.3. Entrainment by light and temperature
Transfer of flies from continuous darkness to a light cycle leads to entrainment of the
activity rhythm. The process of entrainment involves a change of T from its free-running value
in darkness (TDD) to the period of the light-dark cycle or Zeitgeber; in the example shown (Fig.
5) to the exact 24 hours of LD 16:8. After discontinuation of the light pulses, x returns to a
value close to TDD at the outset. When entrainment has occurred, a steady state phase
relationship (f) to the Zeitgeber is achieved with i|/-values depending, among other things, on
the relative periods of the endogenous rhythm and the environmental light cycle (Pittendrigh,
1981; Saunders, 1982). In the case of the blow fly, which is a diurnal insect, the clock-
controlled locomotor activity becomes confined, in this example, to the 16 hours of light. The
small amount of activity immediately following light-off (arrowed) is an exogenous 'masking'
or 'rebound' effect caused by the abrupt ending of the light.
Timeh
12.00 24.00 12.00 24.00 12.00
Day 1
DD
LD 16:S
DD
Figure 5. Entrainment of the locomotor activity rhythm of a female C vicina, initially in DD at
20°C, to a light-dark cycle (LD 16:8), with a final return to DD; the free-running rhythm
adopts the period of the light cycle (24 hours) during the period of illumination.
The 'mechanics' of entrainment may be explained by summing the phase shifts (advances
and delays) caused by consecutive light pulses until the system achieves its steady state
(Pittendrigh, 1981; Saunders, 1982). The key to this process lies in families of phase response
curves (PRCs) constructed from the phase-shifting effects of single pulses of light of different
duration and irradiance applied, at sequentially later circadian phases, to an otherwise free-
running rhythm of activity. In short, such PRCs generally show phase delays (-Acj)) when the
light pulses fall during the early subjective night (circadian time, Ct 12 to 18), and phase
advances (+A(t)) during the late subjective night (Ct 18 to 24). On the other hand, rather small
(or no) phase shifts occur during the subjective day (Ct 0 to 12), often giving rise to a so-called

'dead zone'. Light pulses of increased irradiance or duration cause larger phase shifts allowing
the rhythm to entrain to a wider range of Zeitgeber periods, and to achieve greater values of |/.
Short or weak light pulses thus give rise to low 'amplitude' (Type 1; Winfree, 1970) PRCs
with small delays and advances (see Fig. 6 for C. vicina exposed to 1 h pulses of light). Longer
or brighter light pulses may give rise to high 'amplitude' Type 0 PRCs with delays and
advances approaching one half of the circadian cycle.
+10r
+4
+2
-2
-8
-10'-
_LD_Da_S-j
12 18 24 12
Figure 6. Phase response curve (PRC) for the locomotor activity rhythm of C vicina exposed
to 1 hour pulses of white light, showing phase delays (-Acj) in hours) and phase advances (+A(t))
as afimctionof circadian phase (in hours)(y axis). Data are double-plotted (• and D).
Pittendrigh's entrainment model may also be used to calculate ranges of entrainment to
Zeitgeber cycles (T) that are multiples (i.e. T -- 48, 72 or 96 hours) or sub-multiples (T ~ 12
hours) of T. Such calculations have proved to be crucial to unravelling entrainment to Nanda-
Hamner protocols (see below), important in the analysis of the role of circadian oscillations in
photoperiodic time measurement (Saunders, 1978).
Insect circadian rhythms also entrain to temperature cycles, or thermoperiods. However,
unlike light, temperature cycles differ in that the separate phase shifting effects of temperature
steps-up and steps-down may be studied. This cannot be done with light because light-on
automatically leads to a period of higher light intensity, which frequently causes arrhythmicity
(Hong and Saunders, 1994), in which 'phase' has no clear meaning. Using protocols
established by Zimmerman et al. (1968) for the rhythm of pupal eclosion in Drosophila
pseudoobscura, Saimders and Hong (2000) used separate temperature steps-up (15 to 20°C)
and down (20 to 15°C) to simulate phase shifting by a low temperature pwte lasting 6 hours.
Computation of a theoretical PRC for 6-hour low temperature pulses, led to a prediction of
steady state entrainment of the activity rhythm to a thermoperiodic cycle of 18 h at 20° and 6 h
at 15°C. This phase relationship showed that activity was confined to the daily thermophase,
as expected for a diumally active insect.
The basic properties of the circadian system, as outlined above, have proved to be essential
in understanding time measurement in the photoperiodic clock.

3. PHOTOPERIODIC TIMING
3.1. The photoperiodic response
In common with many other insects inhabiting northern latitudes, the blow fly responds to
seasonal changes in day length by producing a dormant or diapausing stage as winter
approaches. In C vicina, the diapause stage is the third instar larva, but the stage sensitive to
photoperiodic change is maternal. Thus, female flies exposed to the long days (or short nights)
of summer lay eggs giving rise to larvae that develop without arrest. On the other hand, those
experiencing autumnal short days (or long nights) lay eggs giving rise to larvae that enter
diapause as post-feeding larvae, after burrowing into the soil, but before puparium formation
(Saunders, 1987). Flies may be diverted from the diapause 'pathway', however, by
temperatures in the larval habitat above about 15°C (Vaz Nunes and Saunders, 1989) or by
larval over-crowding (Saunders, 1997).
Many insects entering a winter diapause acquire a range of physiological and behavioural
characteristics referred to as the 'diapause syndrome'. This may include reduced metabolism,
an increased storage of metabolites, especially fat, augmentation of epicuticular waxes, and an
increased cold tolerance. In C vicina, there is no significant increase in lipid storage
(Saunders, 1997, 2000a), but they do acquire an increased cold tolerance - particularly in
strains from northern latitudes - which might be an integral part of the diapause syndrome
(Saunders and Hayward, 1998). The immediate cause of diapause is an alteration to the
endocrine control of moulting and metamorphosis (Denlinger, 1985; Saunders, 2000b). In the
case of C vicina it involves a 'block' to the release of the neuropeptide prothoracicotrophic
hormone (FTTH) from the brain and an associated low titre of haemolymph ecdysteroids
(Richard and Saunders, 1987). It is clear, however, that diapause is not merely a cessation of
activity: it is an actively induced and alternative developmental pathway regulated by the
expression of a set of characteristic diapause-related genes in the brain (Denlinger et al., 1995).
Since the induction of larval diapause in C. vicina is of maternal origin, it is clear that the
photoperiodic clock and the clock controlling the overt rhythm of locomotor activity (see
above) are operating concurrently in the female fly. This raises an interesting question: are the
two clocks 'the same' or 'different'? This question will be addressed in a later section.
3.2. The photoperiodic response curve
The photoperiodic responses of an insect such as C vicina are best described by a
photoperiodic response curve (PPRC) constructed by exposing a series of groups of the insect
to stationary light cycles during their sensitive period. The PPRC then plots diapause incidence
as a function of photophase. A typical curve for C vicina, like most 'long day' or summer
active insects, shows low diapause incidence under long days, a high incidence under short
days, and a sharp discontinuity (the critical day or night length) between the two (Fig. 7). The
ecologically important critical day length (CDL) separates diapause-inducing from diapause-
averting photoperiods. CDL is also a function of latitude, more northerly populations having a
longer CDL than populations to the south. Low temperature may affect the PPRC by raising
diapause incidence under strong short days, whereas high temperatures may lower it, although
different constant temperatures may have rather little affect on the value of the CDL. On the
other hand, thermoperiod may have diapause inducing/averting affects in a similar fashion to
photoperiod (Saunders, 1982).

100
20 24
8 12 16
Photoperiod, h/24
Figure 7. Photoperiodic response curve (PPRC) for the production of diapause larvae by
females of C vicina exposed to a range of photoperiods at 20°C. The critical day length (50%
response) is at LD 14.5:8.5.
3.3. The circadian basis of photoperiodic time measurement
Erwin Running (1936) was the first to suggest that seasonal photoperiodic timing had its
origin in the circadian system. Twenty years later, Colin Pittendrigh (1960, 1972) developed
these ideas into a number of more specific models based on circadian entrainment. The
literature and the evidence for this apparently causal association has been reviewed many times
and will not be repeated here. For tiie early literature, see Saunders (1982); Vaz Nunes and
Saunders (1999) provide a more recent review.
The covert operation of the circadian system in photoperiodic time measurement (PPTM) is
revealed by several experimental procedures, the most widely used being the Nanda-Hamner
protocol. In this type of experiment, insects are exposed to a series of non-24 hour light-dark
cycles in which the light phase is held constant but darkness systematically extended through
several multiples of T (e.g. L = 12 hours; D = 8 to 72 hours). In many insects, including C
vicina (Fig. 8), diapause incidence rises and falls with a circadian periodicity. It is high when
the overall light-dark cycle (T) is close to 24, 48 or 72 hours (i.e. when T = modulo T) but low
when T is close to 36, 60 or 84 hours (T = modulo T + Yii). The interval between peaks reflects
the circadian period (T). Other procedures, which will not be reviewed here, also reveal a
covert rhythmicity in PPTM. These include (1) interruption of the extended night of a long
cycle (e.g. LD 12:60) by systematically later short pulses of light (the Biinsow protocol), and
(2) the powerful use of skeleton photoperiods in the 'zone of bistability' (Pittendrigh, 1966;
Saunders, 1978; Vaz Nunes and Saunders, 1999).
These experiments demonstrate that circadian rhythmicity is involved in PPTM, but tell us
little about the exact nature of that involvement. This uncertainty has given rise to a number of
models for time measurement, details of which are given by Vaz Nunes and Saunders (1999).

100|
c
0 8 0 I
a
<i;6oi
0.401
20
-•-•- : : i . = . = ^ .
12 24 36 48 60 72 84
Duration of light/dark cycle, T (h)
Figure 8. Nanda-Hamner experiment. Production of diapausing larvae by females of C vicina
exposed (at 20°C) to a range of light cycles each containing a 13 hour light component and a
variable (7 to 67 hour) scotophase to give LD cycles (T) between 20 and 80 hours. Peaks of
high diapause incidence occurred at about 24 hour intervals equivalent to multiples (x, 2T and
3T) of the circadian period.
The 'double circadian oscillator' model (Vaz Nunes, 1998), v^th two oscillators separately
measuring long and short nights, currently offers a good simulation of the phenomenon.
'Positive' Nanda-Hamner responses have now been recorded in about 12 species of insects and
a mite. 'Negative' responses, lacking the obvious periodicity in diapause response, have
however been recorded in a further 11 species. These apparently negative responses are
frequently identified as evidence for a non-circadian or 'hourglass' type of clock. However,
Lewis and Saunders (1987) and Vaz Nunes and Saunders (1999) conclude that hourglass-like
and oscillatory timers are both based on the circadian system, the former showing heavy
damping in extended periods of darkness and the latter more persistence. Even the 'classical'
example of an hourglass type of photoperiodic clock - that in the aphid Megoura viciae (Lees,
1973) - has now been shown to have a circadian basis (Vaz Nunes and Hardie, 1993).
4. THE MULTIOSCILLATOR CIRCADIAN SYSTEM
Circadian rhythmicity is essentially a cellular phenomenon. Nevertheless, apparently
independent or semi-independent, light entrainable circadian clocks exist at all levels of
organisation from cells, through tissues to organs. Such systems have been described, inter
alia, in nervous tissue, endocrine glands, gonads, malpighian tubules and epidermis (see
Giebultowicz, 1999). Some of these oscillatory components may be truly independent; others
may be part of a physiological hierarchy. There seems little doubt, however, that an insect
represents a muhioscillator circadian 'system'.
Even within the central nervous system that - unsurprisingly perhaps - houses circadian
'clocks' responsible for overt behavioural rhythms, pacemakers are of a muhioscillator
construction. Cells responsible for regulating locomotor rhythmicity in the finit fly Drosophila
melanogaster, for example, have been identified as the so-called lateral neurons (LNs) in the
central brain (Helfrich-Forster et al., 1998). In the mutant disconnected most flies lack these

10
crucial LNs and are totally arrhythmic. However, flies retaining just a single such neuron may
continue to express a rhythm. Possibly homologous cells, that may also be photoreceptive,
have been found in the brain of C vicina (Cymborowski and Korf, 1995).
Adult females of C vicina, as noted above, express locomotor rhythms and measure
photoperiodic time simultaneously. Although the circadian pacemakers for both are probably
located in the brain, and use a photoreceptive input that can by-pass the compound eyes and
ocelli (Cymborowski et al., 1994; Saunders and Cymborowski, 1996), the two systems show
quite different properties that suggest they are 'separate' parts of the multioscillator system.
Locomotor rhythmicity, for example, is regulated by a self-sustaining oscillator that free-runs
in darkness for life at an average period of about 22.5 hours (Kenny and Saunders, 1991).
Photoperiodic induction, on the other hand, is regulated by a circadian system that expresses
(through Nanda-Hamner experiments) an endogenous periodicity much closer to 24 hours, and
bears all the hallmarks of a damped oscillation (Lewis and Saunders, 1987; Saunders, 1998). A
similar separation of locomotor rhythmicity and photoperiodic time measurement is apparent in
D. melanogaster where the latter is retained in behaviourally arrhythmic flies lacking the
period gQiiQ (Saunders et al, 1989; Saunders, 1990).
5. UNRESOLVED PROBLEMS: What we know, and what we need to know
5.L The current molecular model for circadian rhythmicity
In D. melanogaster, a molecular model for the generation of circadian rhythms has
emerged from a welter of studies that suggest overall negative feedback loops involving the
transcription and translation of so-called 'clock' genes (period, timeless and others). In brief,
the widely accepted, or 'orthodox' view starts with the transcription ofper and tim. Two other
genes are involved at this stage: Clock and Email. Their products are thought to form a dimer
that binds to the E-boxes of per and tim to stimulate their transcription. In the cytoplasm the
PER and TIM proteins form ^eir own complex - which is variously delayed by the binding of
PER with the product of another gene, doubletime - and this PER/TIM heterodimer later enters
the nucleus during a specific 'gate' during the subjective night (Ct 19-20). The nuclear
complex then binds to Clock and Email thereby removing the transcriptional activation of per
and tim. Consequently, the PER/TIM dimer degenerates, transcription of per and tim restarts,
and the cycle continues. According to this orthodox view, important time delays in the loop are
brought about by the action of doubletime. Phase shifts, leading to entrainment, are caused by
the action of li^t on TIM. Early in the subjective night, photic degeneration of TIM causes
retardation of nuclear entry and hence phase delay. Later in the cycle, nuclear degeneration of
TIM leads to a phase advance.
5.2. 'Clock' genes and the regulation of circadian and other periods
In D. melanogaster, the genes mentioned above play a clearly important role in the
generation of curcadian rhythmicity. But are they dedicated clock genes? Even in the fiiiit fly,
the proposed feedback loop does not occur in all tissues: in the ovary, for example, PER
remains cytoplasmic, not entering the nucleus to complete a loop. In other insects such as the
house fly, Musca domestica, and the silkmoth, Antheraea pernyi, a similar lack of nuclear
entry has been described, this time in brain neurons which are thought to be pacemaker cells.
In D. melanogaster, fh^per gene is also involved in the generation of an ultradian 'love-song'
rhythm which cycles with a period close to a minute. An ultradian period that short cannot be
explained by a transcription-translation loop involving nuclear-cytoplasmic interactions
because it is far too rapid. At the other end of the scale, the mind simply boggles at how lunar

11
and circannual rhythms - with properties similar to circadian rhythms, but differing only in
period - may be generated. It is almost as though per (and perhaps the other genes) had (and
now have) another, more ancient, role, and have been hijacked to serve as part of a clock loop.
Lastly, there may be clock loops not containing per. In D. melanogaster there are several
reports of admittedly weak behavioural rhythmicity persisting in p^r null mutants (Dowse et
al, 1987; Helfrich and Engelmann, 1987; McCabe and Biriey, 1998). In the photoperiodic
induction of diapause in D. melanogaster, clearly a product of the circadian system (Saunders,
1990), time measurement proceeds, albeit with an altered critical day length, in behaviourally
arrhythmic/7^r^ flies and in flies {per) entirely lacking the period locus. These studies suggest
that the period gene is not absolutely essential, or that other cyclically transcribed genes exist
which may induce some rhythmicity.
The importance of time delays to generate a near-24 hour periodicity has been recognised
for a long time (e.g. Lewis and Saunders, 1987). The orthodox molecular model outlined above
attributes such time delays to interactions with doubletime or delays in nuclear re-entry. It is
difficult to see, however, how such events may be reconciled with the period-associated
phenotypes of the original period mutants {per^ ,1^19 hours; per^, x ~ 29 hours) (Konopka
and Benzer, 1971). Another view is that at least some of the variation in x arises from the
multioscillator construction of the fly's circadian system, i.e. in coupling between individual
cellular oscillators among the lateral neurons. Tight coupling might result in a shorter period,
whereas looser coupling might give rise to a longer period. Unpublished data for C vicina
suggest that such a relationship does exist: in flies with a short x the rhythm of locomotor
activity is more 'precise', whereas as x increases the rhythm 'loosens', eventually to become
arrhythmic, presumably because of very weak coupling between the 'clock' cells.
5.3. Temperature compensation
Problems also exist with the generation of temperature compensation of the circadian
period, one of the defining properties of a 'clock'. For D. melanogaster, one interesting
suggestion is that period stability at different temperatures was related to the length of the
threonine-glycine (T-G) encoding repeat within the 'clock' gQnQ, period. Sawyer et al (1997)
showed that the two major variants, T-G 17 and T-G 20, were distributed in a significant
latitudinal cline in Europe and North Africa, with longer sequence flies predominating to the
north. The length of the T-G repeat was related to the flies' ability to maintain their circadian
period at different temperatures. The association was made plausible by the observation that
per^ flies 'rescued' withper lacking a T-G repeat sequence became behaviourally rhythmic but
lacked temperature compensation (Ewer et al., 1990). It is difficult, however, to see a universal
mechanism in this phenomenon, particularly since in some non-drosophilids the number of T-
G repeats is stable (Nielsen at al., 1994). In Lucilia cuprina, a blow fly relative of C. vicina,
there is no polymorphism, all flies having a T-G doublet.
5.4. The role of circadian rhythmicity in photoperiodism
If there are difficulties in providing a completely satisfactory explanation for the insect
circadian clock, how much more difficult it becomes to unravel the complexities of seasonality.
Photoperiodic induction may be a function of the circadian system, but this step is only one
part of a long concatenation. Included in this sequence are: photoreception; measurement of
night length; accumulation of successive long or short nights by a 'counter'; storage of such
information; its transfer from sensitive to responsive stage; and fmally, regulation of the
release/retention of neurohormones controlling the onset of diapause or continuing develop-
ment. The whole sequence may occupy a large part of the entire life cycle, in the case of C.

12
vicina from the adult female fly to the fully developed larva she produces. We have not seen
the end of circadian research.
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Reaktion. Berichte der deutschen botanischen Gesellschaft 53, 590-607.
Cymborowski, B., Korf, H.-W., 1995. Immunocytological demonstration of S-antigen
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the adult locomotor activity rhythm in Calliphora vicina induced by non-steroidal
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Denlinger, D.L., 1985. Hormonal Control of Diapause. In: Kerkut, G.A., Gilbert, L.I. (Eds.)
Comprehensive Insect Physiology, Biochemistry and Pharmacology. Pergamon Press
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297.
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Drosophila melanogaster. Behavioral Genetics 17, 19-35.
Ewer, J., Hamblen-Coyle, M., Rosbash, M., Hall, J.C, 1990. Requirement forperiod gQne
expression in the adult and not during development for the locomotor activity rhythms of
imaginal Drosophila melanogaster. Journal of Neurogenetics 7, 31-73.
Giebultowicz, J.M., 1999. Insect circadian clocks: is it all in their heads? Journal of Insect
Physiology 45, 791-800.
Helfrich, C, Engelmann, W., 1987. Evidences for circadian rhythmicity in XhQper^ mutant of
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Helfrich-Foerster, C, Stengl, M., Homberg, U., 1998. Organization of the circadian system in
insects. Chronobiology International 15, 567-594.
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activity in the blow fly, Ccalliphora vicina. Physiological Entomology 19, 319-324.
Hong, S-F., Saunders, D.S., 1998. Internal desynchronisation of the circadian locomotor
rhythm of the blow fly, Calliphora vicina, as evidence for the involvement of a complex
pacemaker. Biological Rhythm Research 29, 387-396.
Kenny, N.A.P., Saunders, D.S., 1991. Adult locomotor rhythmicity as "hands" of the maternal
photoperiodic clock regulating larval diapause in the blowfly, Calliphora vicina. Journal
of Biological Rhythms 6, 217-233.
Konopka, R., Benzer, S., 1971. Clock mutants of Drosophila melanogaster. Proceedings of the
National Academy of Sciences, U.S.A. 68, 2112-2116.
Lees, A.D., 1973. Photoperiodic time measurement in the aphid Megoura viciae. Journal of
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13
theoretical Biology 128, 47-59.
McCabe, C, Birley, A., 1998. Oviposition in theperiod genotypes of Drosophila melano-
gaster. Chronobiology International 15, 119-133.
Nielsen, J., Peixoto, A.A., Piccin, A., Costa. R., Kyriacou, C.P., Chalmers D., 1994. Big flies,
small repeats: The Thr-Gly repeat region on thQ period gene in Diptera. Molecular Biology
and Evolution 11, 839-853.
Pittendrigh, C.S., 1960. Circadian rhythms and the circadian organization of living systems.
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Pittendrigh, C.S., 1966. The circadian oscillation in Drosophilapseudoobscura pupae: a model
for the photoperiodic clock. Zeitschrift far Pflanzenphysiologie 54, 275-307.
Pittendrigh, C.S., 1972. Circadian surfaces and the diversity of possible roles of circadian
organization in photoperiodic induction. Proceedings of the National Academy of
Sciences, U.S.A. 69, 2734-2737.
Pittendrigh, C.S., 1981. Circadian Systems: Entrainment. In: Aschoff, J. (Ed.) Handbook of
Behavioral Neurobiology, vol 4 Biological Rhythms. Plenum Press, New York. 95-124.
Richard, D.S., Saunders, D.S., 1987. Prothoracic gland function in diapause and non-diapause
Sarcophaga argyrostoma and Calliphora vicina. Journal of Insect Physiology 33, 385-392.
Saunders, D.S., 1978. An experimental and theoretical analysis of photoperiodic induction in
the flesh-fly, Sarcophaga argyrostoma. Journal of Comparative Physiology 124, 75-95.
Saunders, D.S., 1982. Insect Clocks, second edition. Pergamon Press, Oxford, pp 409.
Saunders, D.S., 1987. Maternal influence on the incidence and duration of larval diapause
Calliphora vicina. Physiological Entomology 12, 331-338.
Saunders, D.S., 1990. The circadian basis of ovarian diapause regulation in Drosophila
melanogaster: is thQ period gene causally involved in photoperiodic time measurement?
Journal of Biological Rhythms 5, 315-331.
Saunders, D.S., 1997. Under-sized larvae from short-day adults of the blowfly,Calliphora
vicina, side-step the diapause programme. Physiological Entomology 22, 249-255.
Saunders, D.S., 1998. Insect circadian rhythms and photoperiodism. Invertebrate Neuroscience
3, 155-164.
Saunders, D.S., 2000a. Chapter 6, Arthropoda - Insecta: Diapause. In: A. Dom (Ed) Progress
in Developmental Endocrinology, Vol X Reproductive Biology of Invertebrates. (Ed.
Adiyodi) Oxford & IBH Publishing Co. Pvt. Ltd., New Delhi, India.
Saunders, D.S., 2000b. Larval diapause duration and fat metabolism in three geographical
strains of the blow fly, Calliphora vicina. Journal of Insect Physiology 46, 509-517.
Saunders, D.S., Cymborowski, B., 1996.Removal of optic lobes of aduh blowflies(Calliphora
vicina) leaves photoperiodic induction of larval diapause intact. Journal of Insect
Physiology 42, 807-811.
Saunders, D.S., Henrich, V.C., Gilbert, L.I., 1989. Induction of diapause in Drosophila
melanogaster: photoperiodic regulation and the impact of arrhythmic clock mutations on
time measurement. Proceedings of the National Academy of Sciences, U.S.A. 86, 3748-
3752.
Saunders, D.S., Hong, S-F., 2000. Effect of temperature and temperature-steps on circadian
locomotor rhythmicity in the blow fly, Calliphora vicina. Journal of Insect Physiology 46,
289-295.
Saunders, D.S., Haywood, S.A.L., 1998. Geographical and diapause-related cold tolerance in
the blow fly, Calliphora vicina. Journal of Insect Physiology 44, 541-551.
Sawyer, L.A., Hennessy, J.M., Peixoto, A.A., Rosato, E., Parkinson, H., Costa R., Kyriacou,
C.P., 1997. Natural variation in a Drosophila clock gene and temperature compensation.

14
Science 278, 2117-2120.
Vaz Nunes, M., 1998. A double circadian oscillator model for quantitative photoperiodic time
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Vaz Nunes, M., Hardie, J., 1993. Circadian rhythmicity is involved in photoperiodic time
measurement in the aphid Megoura viciae. Experientia 49, 711-713.
Vaz Nunes, M., Saunders, D.S., 1989. The effect of larval temperature and photoperiod on the
incidence of larval diapause in the blowfly, Calliphora vicina Physiological Entomology
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Vaz Nunes, M., Saunders, D.S., 1999. Photoperiodic time measurement in Insects: A review of
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circadian oscillation in Drosophilapseudoobscura and its entrainment by temperature
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©2001 Elsevier Science B.V. All rights reserved. 15
Molecular control of Drosophila circadian rhythms
Peter Schotland and Amita Sehgal
Department of Neuroscience, University of Pennsylvania, 232 Stenimler Hall, Philadelphia,
PA 19104, USA
The ubiquity of circadian rhythms testifies to their adaptive significance. Studies in
Drosophila melanogaster have greatly advanced our understanding of circadian rhythms at
the molecular level. To date, at least seven genes in Drosophila have been described and
shown to participate in transcription-translation based feedback loops that comprise the
central oscillator controlling overt circadian behaviors. Many of these genes have functional
homologues in mammals. There has also been recent progress in describing, at the molecular
level, how temporal information from the environment is conveyed to the central oscillator
and, how, in turn, that information is conveyed to the rest of the organism.
1. INTRODUCTION
Circadian rhythms are displayed by organisms ranging across the phyla from
unicellular cyanobacteria to multicellular fungi, plants and higher order mammals, including
humans (Dunlap, 1999). The universality of circadian rhythms attests to their adaptive
significance. Circadian rhythms are characterized by the same three fundamental properties
in different organisms, which presumably allows them to serve similar adaptive functions
(Pittendrigh, 1960). 1. They can be synchronized, or entrained, by environmental stimuli
called zeitgebers (German for "time givers")- The dominant zeitgebers are, not surprisingly,
light and temperature as they follow closely the earth's daily rotation. 2. They persist, or
freerun, under constant conditions; i.e., after removal of the entraining zeitgeber. Under these
conditions the length of a complete cycle (the period) is usually between 23 and 25hr (rarely
the 24hr rhythm of the zeitgeber); hence, the term "circa" (about). The freerunning period also
varies somewhat between individuals. 3. The period of a circadian rhythm shows little
variance with respect to (physiological) temperature, and is said to be temperature-
compensated. Note that the compensation of period with temperature does not preclude
temperature as a zeitgeber. Indeed, competition experiments in Neurospora crassa have
shown temperature to be dominant over light (Liu et al., 1998). The phase of the rhythm is
often temperature dependent, however.
Chronobiologists like to think heuristically in terms of a circadian system that contains
a central clock, an input pathway and an output pathway. The input pathway conveys time of
day information from the environment to the clock, and the output pathway conveys the

16
internal time from the clock to other systems to allow for the temporal organization of
behavior and physiology. Although input and output are often thought of as independent,
linear pathways, this is probably an over-simplification of the system. It is more likely that
the three components of the circadian system affect one another and, as is evident from the
molecular analysis, have a significant amount of overlap. Indeed, some molecules appear to
play a role in all three components of the system. There is also evidence suggesting that in
some organisms there is no central oscillator, but rather several "peripheral" oscillators that
can function (even entrain) independently of one another. To further complicate matters,
some of the feedback mechanisms that are characteristic of the clock are sometimes also
found in the input and output pathways. Nevertheless, a set of criteria was proposed that
would facilitate the identification of clock components, i.e. those molecules that play a role in
the time keeping mechanism (Zatz, 1992). Clock components that actually provide time cues,
usually through oscillations of their abundance or activity, are called state variables.
The ubiquity of circadian rhythms provided researchers with the hope that, if the
underlying mechanisms are conserved, dissection of those mechanisms in simple organisms
would lead to an understanding of rhythms in higher organisms. Indeed, the molecular
mechanism of the clock in most organisms studied appears to be conserved and turns out to be
a transcription-translation based feedback loop comprised of cycling RNAs and proteins.
While most of this characterization has been done in the bread mold, Neurospora crassa, and
the fruit fly, Drosophila melanogaster, aspects of it have been described in mammals and in
cyanaobacteria (Dunlap, 1999).
2. CIRCADIAN RHYTHMS IN DROSOPHILA
Since the pioneering work of Ron Konopka in 1971 (Konopka, 1971), more than 15
clock and clock controlled genes were identified in Drosophila (Dunlap, 1999). Indeed,
Drosophila proved to be a fruitful system for the study of circadian rhythms long before the
advantages of genetics came into play. Pittendrigh (Pittendrigh, 1960; Pittendrigh, 1967)
demonstrated the existence of quantitative, well defined circadian behaviors in Drosophila
pseudoobscura and developed assays still used to elucidate fundamental properties of the
clock, including the discovery of the molecules discussed in this review. Using selective
breeding experiments with Drosophila pseudoobscura, Pittendrigh demonstrated the
heritability, and, hence, the genetic basis of certain clock characteristics such as period length
and phase, but the focus of genetic research soon moved to Drosophila melanogaster because
of the powerful genetic technologies being developed in that species.
Genetic studies in Drosophila have predominantly exploited two overt rhythms-
locomotor activity and eclosion. When individual flies are placed in a glass tube with a little
food, their locomotor activity can be monitored by counting the frequency of crossing of an
infrared beam. When maintained in 24hr lightidark cycles, flies display a bimodal
distribution of locomotor activity with peak activity occurring in late night/early morning and
late day/early evening (Hamblen-Coyle, 1992). When placed in freerun, these peaks merge to
a single bout of activity during the subjective day. These daily bouts of activity will persist in
constant conditions, with no reference to external time, for the lifetime of the fly- as long as
two months. It is extraordinary that the same holds true for many mammals, with mice and
hamsters able to display circadianly gated behavior under constant conditions for years at a

17
stretch. Eclosion, the hatching of pharate adults from their pupal casings, is regulated by the
clock such that it occurs during a few hours near dawn (Pittendrigh, 1967). Eclosion and
pupal development are independently regulated such that if pupal development is completed
after dawn, eclosion is delayed several hours until the next dawn (Qiu, 1996). Hence,
eclosion is said to be circadianly gated.
Circadian rhythmicity has been detected in other systems such as visual and olfactory
sensitivity, feeding, and oviposition (Chen et al., 1992; Krishnan, 1999; McCabe & Birley,
1998). Although these clock regulated phenomena have yet to be useful in elucidating the
underlying molecular clockwork, they have been shown to be regulated by the molecules
discovered studying eclosion and locomotor activity.
2.1. per, dm and the feedback loop
The first systematic effort to isolate circadian rhythm mutants was done by Ron
Konopka in 1971 (Konopka, 1971). The result, three alleles mapping to the period locus
(per), is perhaps the most important in the molecular analysis of circadian rhythms.
Konopka's genetic screen found pretty much every circadian mutant conceivable: a short
period mutant, pe/^""^ ipe/ that reduced freerunning period to 19hr, a long period mutant,
per^^^^ (per^), with a freerunning period of 29hr, and a mutation producing total arrhythmia,
per^ The per mutants affected both eclosion and locomotor activity implicating a role for
per in the central clock. That single nucleotide mutations in one gene could affect a complex
behavior in a variety of ways was an important discovery not only for the field of circadian
rhythms, but for behavioral studies in general. For many years, the period locus was
considered the paradigm for a behavioral gene because only behavioral defects were found in
flies carrying the per mutations; i.e., the flies were developmentally and anatomically normal.
Recently, however, mutations affecting the clock have been found in genes with roles in other
processes including embryonic development (discussed below).
Several additional alleles of per are now known (Hamblen et al., 1998; Konopka et al.,
1994). per remained the only known bona fide clock gene until the isolation of the timeless
(tim) mutation in 1994 (Sehgal et al., 1994). tim is a null mutation producing total
arrhythmia. Since then, long and short period alleles of timeless have also been isolated. A
32 amino acid deletion in the N-terminal region of the tim protein increases freerunning
period to 30-48hr (Ousley et al., 1998), the tim^'^ (Matsumoto et al., 1999) allele increases
period to 27 hours and the r/m"'''^"'''"^ allele produces 33hr rhythms (Rothenfluh et al., 2000).
tim^^ shortens period by only 0.5 hours in a wild type background, but it can suppress the per'
long-period phenotype by several hours (Rutila et al., 1996). Recently, 2 short (21-22hr) and
4 long (26-28hr) period alleles of tim have been isolated (Rothenfluh, 2000).
Unfortunately, very little information about the biochemical function of the per and
tim proteins could be inferred from their molecular sequences. The tim sequence is entirely
unique (Myers et al., 1995), and the only protein motif found in per is a protein-protein
interaction domain known as a PAS domain, named for the proteins it was first found inper,
single-minded, and the aryl hydrocarbon receptor nuclear translocator. PAS domains have
since been found in other clock molecules, photoreceptors, developmental proteins and
proteins involved in hypoxia and may confer on many of these proteins the ability to sense
environmental signals (Crews, 1999). Such a function has, however, not yet been described
for the p^r protein.

18
per, tim, vri
elk, CLK, cry
PER
24 hr
Fig. 1. Molecular oscillations of the clock. The abundance of various clock molecules are
plotted over a 24hr, 12:12, light:dark cycle. Lower case indicates RNA, uppercase, protein.
Although this plot is for a light:dark cycle, the gene products behave the same in constant
darkness with two exceptions: 1. the TIM profile more closely follows PER in constant
darkness as light apparently turns over TIM more rapidly than the clock at the beginning of
the day. 2. CRY does not cycle in constant darkness but instead accumulates in a non-
decreasing manner.
Although their biochemical properties were not obvious, analysis of the regulation of
per and tim expression proved to be very informative. Measurements of the RNA and protein
products of both clock genes showed that, keeping with the notion of a state variable (Zatz,
1992), oscillating gene products constitute the core of the Drosophila clock (see Fig. 1). Both
genes encode RNAs that cycle with a circadian rhythm, such that RNA levels are high at the
end of the day/beginning of the night (Hardin et al., 1990; Sehgal et al., 1994; Sehgal et al.,
1995). The per and tim proteins (PER and TIM) also cycle and begin accumulating in the
early evening. PER and TIM bind one another to form heterodimers that are then transported
into the nucleus. Each protein is required for nuclear transport of the other; i.e., in per and tim
null mutants, TIM and PER, respectively, are restricted to the cytoplasm (Hunter-Ensor et al.,
1996; Myers et al., 1996; Saez & Young, 1996; Vosshall et al., 1994). The abundance of both
proteins peaks in the middle of the night (Hunter-Ensor et al., 1996; Myers et al., 1996; Zeng
et al., 1996).

19
per tim
Per Tim
t
PER TIM
DBT
Proteasome
B DAY NKHIT
TRI
l-.-to.s
Fig. 2. The molecular feedback loop in Drosophila. A The Drosophila clock is comprised
of interiocking feedback loops. The per, tim feedback loop is indicated in gray, the dClock
loop indicated in black. Arrows indicate activation, squares inhibition. Also shown are the
dbt dependent turnover of PER via phosphorylation, and the phosphorylation and ubiquitin
dependent degradation of TIM by light. CRY likely plays a role in this process as well. B
During the day, TIM, and, therefore, PER levels are low and insufficient to inhibit CLOCK-
CYC mediated transcription. At night, TIM and PER can rise to levels sufficient to inhibit
CLOCK-CYC.

20
Thereafter, PER levels remain high until the early morning while TIM levels drop off at the
end of the night. TIM and PER are also cyclically phosphorylated, and peak phosphorylation
occurs at the end of the night (Edery et al., 1994; Zeng et al., 1996). Phosphorylation
probably serves many functions, one of which is to target the proteins for degradation.
Phosphorylation of PER by a homologue of casein kinasele encoded by the doubletime (dbt)
locus renders it unstable in the absence of TIM (Kloss et al., 1998; Price et al., 1998).
Consistent with the notion of a state variable, the oscillations described above persist under
constant conditions.
Both TIM and PER are required for cyclic expression of their mRNA's (Hardin et al.,
1990; Sehgal et al., 1995). Since RNA levels are low when protein levels are high a negative
feedback loop model was proposed in which PER and TIM enter the nucleus to inhibit
transcription from the per and tint loci (Hardin et al., 1990) (see Fig. 2A). In support of the
negative feedback hypothesis, overexpression of PER from the rhodopsin promoter (in
photoreceptor cells) reduces levels of the endogenous per transcript (Zeng et al., 1994).
Essential for the maintenance of such a loop is the separation of the phase of RNA synthesis
from RNA inhibition (Dunlap, 1996). This separation may be achieved by the 6 hr lag
between peak RNA and peak protein levels, the regulation of the time of nuclear entry of the
heterodimer, or possibly temporally gated phosphorylation of PER/TIM. The rate of PER
accumulation is dependent on DBT mediated destabilization of PER (Price et al., 1998).
It should be stressed, however, that this is a simplistic view and does not explain all
effects of PER-TIM on their RNA levels. The negative feedback model would predict that
per and tim RNA would be expressed at peak levels in flies where feedback doesn't occur
because of the absence of either protein. However, per^^ and tirn^^ flies express intermediate
levels of per and tim RNA (Hardin et al., 1990; Qiu & Hardin, 1996; Sehgal et al., 1994;
Sehgal et al., 1995). Transcription rates of per and tim are also intermediate in per^^ and tim^^
flies (So & Rosbash, 1997). This suggests that, in addition to negative feedback, the PER and
TIM proteins, either directly or indirectly, also have a positive effect on the expression of the
their RNAs. This positive effect derives, at least in part, from the stimulation of Clk gene
expression by PER-TM (see below)(Bae et al., 1998; Glossop et al., 1999). In addition, TIM
increases levels ofper RNA through a post-transcriptional mechanism (Suri et al., 1999).
Because both PER and TIM lack conventional DNA binding domains and have never
been shown to associate directly with DNA, models for PER action postulated that PER
associates with transcriptional activators and sequesters them, thereby preventing them from
activating transcription (Huang et al., 1993). The per PAS domain, a protein-protein
interaction domain also found in several transcription factors, was hypothesized to mediate
this interaction (Huang et al., 1993). Transcriptional activators of per Sind tim were recently
described and, as predicted, they contain PAS domains as well as bHLH motifs that allow
them to bind DNA (AUada et al., 1998; Bae et al., 2000; Darlington et al., 1998; Rutila et al.,
1998). These activators are encoded by the dClock (dClk) and cycle (eye) genes, and flies
mutant at either locus express very low levels of per and tim RNA (Allada et al., 1998; Rutila
et al., 1998). Furthermore, as predicted by the model, cell culture studies show that PER-TIM
act by inhibiting the activity of CLK/CYC (Darlington et al., 1998). The sites in the per and
tim promoters that are recognized by CLK/CYC contain E-boxes (sequence = CACGTG),
promoter elements that are known to be regulated by bHLH proteins. A recent study using a
cell free system (Lee et al., 1999) demonstrated that CLK and CYC can bind the per promoter

21
E-box together as a heterodimer, but not individually. PER and/or TIM were capable of
disrupting this binding. The molar ratio of the CLKiCYC heterodimer is unchanged by the
presence of PER or TIM, indicating that the probable mechanism of transcriptional inhibition
consists of blocking DNA-association of the heterodimer rather than disrupting the
heterodimer itself (see Fig. 2B).
dClk RNA also cycles, antiphase to per and tim RNA (see Fig. 1) (Bae et al., 1998;
Darlington et al., 1998). dClk RNA is high in the dClk null mutant compared to wild type
trough levels, indicating that dClk gene products also participate in their own negative
feedback loop. Additionally, dClk RNA levels are low in per^^ and tim^^ flies, indicating that
not only does dClk positively regulate per and tim, but per and tim, directly or indirectly, are
positive regulators of dClk. Thus, per, tim, and dClk are engaged in interlocked feedback
loops (Glossop et al., 1999) (see Fig. 2A).
The most recently identified component of the Drosophila circadian system, and
possibly of the feedback loop, is the vrille gene (Blau & Young, 1999). Expression of vri
RNA is clock-controlled and cycles in phase with that of per and tim. Overexpression of vn
attenuates oscillations of per/tim gene products as well as behavioral rhythms, indicating that
vri can affect clock function in addition to being regulated by the clock (Blau & Young,
1999). However, the circadian phenotype produced by the null mutation is yet to be
described. Homozygous loss of vri results in lethality as does loss of dbt, indicating that both
these genes have other essential functions (Blau & Young, 1999; Price et al., 1998).
It should be mentioned that the phenotypes described above for the different mutants
are based largely upon measurements of rest:activity, and to some extent, eclosion rhythms.
Restiactivity rhythms are produced by a specific group of cells in the Drosophila brain called
lateral neurons (Ewer et ^., 1992; Frisch et al., 1994; Helfrich-Forster, 1998; Kaneko et al.,
2(X)0). Expression studies have focused either on these neurons or, more conmionly, on
assays of whole fly heads where the major source of clock RNA and protein is the
photoreceptor cells of the compound eye. Photoreceptor cells are not required for activity
rhythms and so clock proteins in these cells are thought to constitute an autonomous oscillator
that controls an eye-specific function. Similar autonomous or semi-autonomous oscillators
have been described in other parts of the fly and are the subject of another chapter in this
volume (Giebultowicz, 2000).
2.2. Molecular phenotypes of period altering mutations
Thus far, these have only been described for per, tim and dbt. dbf and dbt^ shorten and
lengthen circadian period by accelerating or delaying, respectively, the cyclic profile of PER
phosphorylation (Price et al., 1998). Presumably, delayed phosphorylation increases stability
of the protein so it stays around longer while accelerated phosphorylation hastens protein
turnover and shortens period. Likewise, the per^ mutation shortens period by accelerating the
daily decay of the protein at the end of each cycle (Marrus, 1996; Siwicki et al., 1988). The
per^ mutation, on the other hand, decreases the strength of the PER-TIM interaction and
delays nuclear entry (Curtin et al., 1995; Gekakis et al., 1995). As a result, period is
lengthened. However, there are caveats to these simplistic models. For instance, one might
expect that a decrease in stability of the protein would also delay its accumulation (given that
the delay in PER appearance relative to its mRNA is due to protein turnover) and thereby
lengthen period and vice versa. There is, in fact, a PER mutant that is hypophosphorylated.

22
shows increased stability, accumulates faster and shortens period (Schotland et al., 2000).
However, the short period phenotype of this mutant may be due to an additional defect in its
ability to effect negative feedback. Clearly, there are many different aspects of protein
regulation and most, if not all likely contribute to the determination of period.
Mutations in dm have similar molecular phenotypes. TIM^^ shows prologed nuclear
localiation and protracted repression of per/tim transcription suggesting that its stability is
increased (Rothenfluh et al., 2000). TIM suppresses PER cycling and reduces overall PER
levels (Matsumoto et al., 1999). These effects on PER are apparently mediated through post-
transcriptional effects on per RNA. Finally, the tim^^ mutation changes the profile of TIM
phosphorylation to somehow rescue the delayed nuclear entry and longer period conferred by
the per^ mutation (Rutila et al., 1996). Since tim^^ does not affect the PER^-TIM interaction,
as measured in yeast, it is thought to be a bypass suppressor (Rutila et al., 1996).
2.3. Mechanisms that entrain the clock to light
If the per and tim gene products are state variables whose levels provide time-of-day
cues to the organism, it follows that resetting of the clock in response to any stimulus would
be accompanied by changes in the levels of these components. The question is which clock
component changes first and mediates the resetting response? In Drosophila, the TIM protein
shows an acute response to light such that it is degraded within 30-90 minutes of light
treatment (based upon the cell type being studied) (Hunter-Ensor et al., 1996; Myers et al.,
1996; Zeng et al., 1996). We now know that TIM is first phosphorylated on tyrosine residues
in response to light, ubiquitinated and then degraded by the proteasome (Naidoo et al., 1999).
A number of lines of evidence support the idea that the TIM response mediates behavioral
resetting: (1) Dose response studies show that light pulses of different intensity produce
corresponding changes in the TIM response and the behavioral response to light (Suri et al.,
1998; Yang et al., 1998). (2) The action spectrum for the TIM response to light matches that
of the behavioral response- both are most sensitive in the blue light region of the spectrum
(Suri et al., 1998). (3) Mutants that affect the TIM response to light also affect behavioral
resetting (Yang et al., 1998). (4) TIM interacts with the circadian photoreceptor,
cryptochrome (CRY), in a light-dependent manner (Ceriani et al., 1999).
Cryptochromes are flavin-binding molecules with homology to DNA photolyases.
They were first identified in plants where they also turn out to have a role in circadian
photoreception (Cashmore et al., 1999). In Drosophila, the only known CRY is essential for
some circadian responses to light such as appropriate behavioral resetting in response to light
pulses (nonparametric entrainment) and arrhythmic locomotor activity in the presence of
constant light (Emery et al., 2000; Stanewsky et al., 1998). However, entrainment to
lightidark cycles can also be mediated, either directly or indirectly, by the visual system
(Stanewsky et al., 1998). It is probably beneficial to the organism to have some redundancy
built into the circadian entrainment system. The role of CRY in TIM degradation has not yet
been reported, but the light-dependent TIM-CRY interaction in cultured cells rapidly blocks
negative feedback by PER-TIM (Ceriani et al., 1999). Presumably this is followed by TIM
degradation which then effects a permanent change in the phase of the clock.

23
ZT19 ZTO ZT3
19°e 25»C 19«C 25 0G ig^C 25«C
PER^-#. ::;::::||||||:|||M||ii^
Fig. 3. PER abundance is temperature dependent. To examine the effect of temperature on
PER abundance Drosophila were maintained at either 19°C or 25®C on a 12:12, lightidark
cycle and collected at hours 19, 0 and 3. Total protein from heads was extracted, western
blotted and stained with an anti-PER antibody. Samples were run in duplicate. PER displays
an increase in temperature at all three temperatures tested.
2.4. Effects of temperature on the clock
Temperature affects the clock in multiple ways. First, temperature sets the limits of
rhythmicity- organisms tend to lose rhythmicity at very high or very low temperatures.
Within this temperature range the period of the rhythm is compensated such that it does not
change. However, the phase of the rhythm can be reset by temperature. As one might
imagine, it is difficult to tease apart the mechanisms involved in these different responses.
Levels of clock components change with the ambient temperature. As demonstrated
by Majerak et al (Majercak et al., 1999), levels of PER are higher at all times at 29°C as
compared to 19°C. We have seen the same result at 19°C vs 25^C (see Fig. 3). Thus, the
protein oscillates around a higher level at the higher temperature. In addition, the daily
protein profile is altered such that the protein accumulates earlier in a lightidark cycle. This is
apparently mediated by a temperature-dependent splicing event in the per RNA (Majercak et
al., 1999). The behavioral consequences of the altered protein profile are that locomotor
activity tends to be concentrated more in the daytime hours at low temperatures and
distributed in the morning and evening hours at higher temperatures (Majercak et al., 1999).
Normally, higher levels of PER result in a shorter period (Baylies et al., 1987; Cooper
et al., 1994; Cote & Brody, 1986; Smith & Konopka, 1981). However, this does not happen
at higher temperatures due to compensation mechanisms that are largely unknown. Although
a number of mutants that affect temperature compensation have been identified and some
mechanisms have been proposed (Hamblen et al., 1998; Huang et al., 1995; Leloup &
Goldbeter, 1997; Price, 1997; Sawyer et al., 1997), there is no consensus on the process
involved. The mutations that affect temperature compensation map to various regions of the
PER protein, suggesting that anything that changes the conformation/folding of the protein
can produce defects in temperature compensation. Finally, the mechanisms underlying
temperature entrainment in Drosophila are not yet understood either.

24
2.5. Output mechanisms
The output pathway remains the least understood aspect of the circadian system.
While inroads have been made into our understanding of the clock itself and entrainment
pathways in Drosophila (at least in response to light, if not temperature), little is known about
how the clock transmits time-of-day cues and produces overt rhythms. This paucity of
information at the output end is true of all organisms where clocks have been studied at a
molecular level. As peripheral oscillators are discussed in the chapter by Giebultowicz et al,
here we will focus on the control of the rest:activity output.
The lateral neurons release a neuropeptide, pigment-dispersing factor (PDF), which is
required for restractivity rhythms (Renn et al., 1999). It may also be required for eclosion
rhythms as these rhythms are disrupted when PDF is overexpressed in specific locations
(Helfrich-Forster et al., 2000). Using antibodies to PDF, arborizations of the axons extended
by lateral neurons have been traced (Helfrich-Forster & Romberg, 1993). They innervate
large regions of the Drosophila optic lobes and also extend to the lateral neurons on the other
side of the brain. However, the nature of the cells innervated is not known nor, for the most
part, are the molecules that act downstream of the clock. Thus, the molecular links between
the clock proteins and PDF and between PDF and the activity rhythm have not been
identified. Although protein kinase A (Levine et al., 1994; Majercak et al., 1997) is known to
be an output molecule required for restractivity rhythms, its location in the output pathway
remains a mystery. Interestingly, CREB (Belvin et al., 1999) which sometimes, but not
always, is downstream of PKA, affects PER protein cycling, but PKA itself does not.
3. CONCLUSION
Along with the recent explosion in our understanding of the molecular underpinnings
of the clock, it has been a delight to see that the work described here is applicable to other
systems, per, tim, dClk, eye, dbt, and ery all have homologues in the mammalian system,
many of them functional counterparts (see the review by Dunlap) (Dunlap, 1999). There are 3
manmialian per homologues that cycle with a circadian rhythm and function as (putative)
negative elements in a feedback loop. Mammalian Cloek and eye (bmall/mop3) can
heterodimerize and activate transcription from E-boxes (Gekakis et al., 1998; Rutila et al.,
1998; Shearman et al., 2000), and tau (mammalian dbt) has been shown to phosphorylate the
mper proteins in vitro (Lx)wrey et al., 2000).
Some important differences between the mammalian and Drosophila systems should
be mentioned. The two known manmialian cry homologues have not been shown to mediate
circadian photoreception but, surprisingly, appear instead to have a role as negative elements
in the feedback loop (Griffin et al., 1999; Kume et al., 1999; Shearman et al., 2000; Vitatema
et al., 1999) and are required for behavioral rhythmicity (van der Horst et al., 1999; Vitatema
et al., 1999). tim's role in the mammalian system is still unknown although, in contrast with
Drosophila, tim is essential for embryonic development (Gotter et al., 2000). Finally, light
sensitivity does not appear to be mediated by a light sensitive protein such as TM, but rather
via photic induction of the mper RNAs (reviewed by Dunlap, 1999).
In addition to the molecular conservation seen in animals, the mechanism of the
transcription-translation based negative feedback loop is found across the phyla in
cyanobacteria, fungi, and plants.

*25
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©2001 Elsevier Science B.V. All rights reserved. 31
Organization of the insect circadian system: spatial and developmental
expression of clock genes in peripheral tissues of Drosophila melanogaster
J. M. Giebultowicz, M. Ivanchenko and T. Vollintine
Oregon State University, Department of Entomology, 2046 Cordley Hall, Corvallis, OR
97331, USA
Insects display circadian rhythms in all aspects of their lives. The term circadian
clock is used to describe the mechanism that generates daily rhythms in insects and other
organisms. Genetic and molecular studies on Drosophila melanogaster revealed that the
clock mechanism comprises a suite of genes regulated via feedback loops w^hich results
in their cycling with a circa 24 h period. Two key clock components, genes period (per)
and timeless (tim), cycle with a peak in the late day followed by a peak in their protein
products PERIOD (PER) and TIMELESS (TIM) during the night. Oscillations of these
clock molecules are found within and outside of the central nervous system. In a
systematic study of fly internal organs, we identified new tissues, such as alimentary tract
and fat body, in which PER and TIM cycle in light/dark and constant conditions implying
the existence of multi-cellular local oscillators. We then used transgenic flies carrying
per-acZ or tim-G¥? reporter constructs to determine the activity of per and tim in
peripheral organs at different stages of metamorphic development. We found that the two
clock genes are tumed on at different metamorphic stages in different tissues. Thus, the
timing of clock gene activation in the periphery is tissue-specific and, therefore, cannot
be accounted for by development-dependent hormonal fluctuations in the hemolymph.
Moreover, at least some peripheral oscillators in adults also appear independent of the
fly's hormonal milieu with regard to their phases. These oscillators are self-sustained and
directly photoreceptive in vitro. Collectively, these observations imply that the fly
circadian system is not organized hierarchically, but rather, includes independently
operating oscillators, synchronized by the external light/dark cycles.
1. INTRODUCTION
Insect life functions are tightly synchronized with the predictable enviroimiental
changes associated with the succession of day and night (Saunders 1982). Such
synchronization is achieved by the circadian system, or clock, which generates daily
rhythms at the biochemical, physiological and behavioral levels. The defining properties
of circadian rhythms (see D. Saunders, this volume) include their entrainment by
enviroimiental cycles and persistence in constant environment with a free-running.

32
temperature compensated period. The molecular mechanism of circadian timing has been
studied vigorously in Drosophila melanogaster, as well as in other organisms, and recent
advances have been summarized in several reviews (Dunlap 1999; Giebultowicz 2000;
Scully and Kay 2000). Briefly, the clock comprises a set of genes controlled through
autoregulatory feedback loops by their proteins, which form heterodimers and enter the
cell nuclei at specific times of the day/night cycle. In Drosophila, proteins dCLOCK and
CYCLE, encoded by dclock (dclk) and cycle (eye) genes, form dCLK-CYC dimers that
activate transcription of two other essential clock genes, period (per) and timeless (tim),
during the day. At the same time, dCLK-CYC dimers inhibit transcription of the dclk
gene. During the late evening, however, PER-TIM dimmers enter the nucleus, and bind
dCLK-CYC complexes, thereby repressing the per and tim genes but releasing the
repression of dclk (Glossop et al. 1999). Recent discovery of similar interdependent
molecular loops in the mammalian (Shearman et al. 2000) and fungal (Lee et al. 2000)
circadian systems suggests that such loops represent a widespread feature of the clock
regulation. It is also known that the phase of the clock oscillations can be reset through a
rapid change in the level of an essential clock component in response to an
environmental signal. For example, in Drosophila, resetting of the clock by light involves
degradation of TIM protein (reviewed in Young 1998).
To understand the organization of the insect circadian system, two important
questions need to be addressed: Which cells in the insect body are bestowed with special
abilities to keep track of time and how are the time-keeping centers synchronized with
each other? One approach to identify timing centers responsible for behavioral rhythms is
monitoring clock outputs after surgical manipulations of the central nervous system
(CNS). Clocks controlling locomotor activity rhythms have been mapped to specific
areas of the optic lobes in cockroaches and crickets (see review by K. Tomioka, this
book). A series of elegant studies in D. melanogaster has demonstrated that the
pacemaking center for the locomotor activity rhythm is a group of cells called lateral
neurons (Helfrich-Forster et al. 1998; Kaneko 1998). In silkmoths, the central brain has
been identified as essential for the rhythms of eclosion and locomotor activity (Truman
1972), however, the rhythm in eclosion-preparatory behavior was found to be brain-
independent (Truman 1984). Some other rhythms also persist independently of the CNS.
For example, the rhythms of cuticle deposition in locusts and sperm release in moths, are
maintained in isolated organs in vitro, indicating that they are driven by local clocks (for
review see Giebultowicz 1999). This data, along with observation of differences in
formal properties of multiple output rhythms in a single organism (Saunders 1986),
provided early evidence that the insect circadian system may have multi-oscillatory
organization with local clocks controlling tissue-specific functions.
2. EXPRESSION OF CLOCK GENES IN PERIPHERAL TISSUES
The cloning of the per gene in Drosophila melanogaster and the generation of
antibodies against PER protein, made it possible to demonstrate the broad distribution of
per mRNA and PER protein within and outside of the CNS (reviewed by Hall 1995).
Cyclic expression of PER was detected in lateral neurons controlling locomotor activity,
in a few other subsets of neurons, and in large groups of glial cells in the brain, as well as
in photoreceptor cells comprising the compound eyes (Siwicki et al. 1988; Zerr et al.

33
u -
A
L_
^ > - TIM , J
- • — PER
• • ^ 1
8 12 16 20 24 4 8 12 16 20 24 4
Time (h)
Figure 1. TIM and PER cycling in Drosophila Malpighian tubules, assayed by
immunofluorescence. Tissues were dissected from flies kept in a normal 12:12 LD
cycle (A), or from flies that were transferred to DD for 2 days (B). White bars indicate
day, black bars indicate night, and hatched bars indicate subjective day (adapted from
Ivanchenko et al., submitted).
1990; Kaneko et al. 1997). Among peripheral tissues, the most complete picture of the
expression patterns of PER and TIM proteins was obtained for the Malpighian tubules
(Hege et al. 1997; Ivanchenko et al. submitted). There are two pairs of Malpighian
tubules in D. melanogaster consisting of excretory epithelium, which is dominated by
large principal cells, all expressing clock molecules. In 12:12 h light-dark cycles (LD),
accumulation of PER and TIM in the nucleus begins simultaneously at Zeitgeber Time
(ZT) 16, consistent with the fact that these proteins enter the cell nuclei as PER-TIM
dimers (Fig. lA). TIM peaks late at night, and then abruptly disappears after lights-on.
PER accumulates in parallel with TIM but persists in the cell nuclei during the beginning
of the light phase. In constant darkness (DD), TIM and PER are also rhythmic, however,
both proteins are present in the cell nuclei for longer periods of time, compared to LD
(Fig. IB). Similar profiles of TIM and PER have been demonstrated in brain neurons and
in whole heads, as determined by immunocytochemistry and Western blotting,
respectively (Marrus et al. 1996; Kaneko et al. 1997).
Although Malpigian tubules are the only peripheral organ in which single cell
resolution was used to demonstrate cycling in both PER and TIM, the presence of per
mRNA and PER protein was reported in many other organs of D. melanogaster (Liu et
al. 1988; Saez and Young 1988; Zerr et al. 1990; Hege et al. 1997; Plautz et al. 1997;
Emery et al. 1997). Recent use of transgenic flies carrying regulatory tim sequences
fused to the Green Fluorescent Protein (r/m-GFP ) demonstrated that tim is co-expressed
with per in several tissues (Kaneko and Hall 2000). However, it was not previously
demonstrated whether both genes show cyclic and free-running expression in any tissue
other than Malpighian tubules. We conducted a systematic survey of peripheral organs
with respect to the expression of clock proteins PER and TIM in LD and free-running
conditions, determining their levels at 6 h intervals. Simultaneous staining of the same

34
Figure 2. Internal organs of D. melanogaster that show cycHc co-expression of PER and
TIM, as assayed by double immunofluorescense. Tissues were dissected at 6 h intervals,
fixed, and reacted with a mixture of anti-PER and anti-TIM antisera at their optimal
concentrations (Giebultowicz and Hege 1997). Immunoreactivity was detected with
Alexa flurophores (Molecular Probes). Images represent nuclear TIM signal at the time
of its peak (ZT22); PER was co-localized in the nuclei of the same cells (not shown). Six
flies were used per time point. Abbreviations: eso, esophagus; pro, proventriculus; hg,
hindgut; mt, Malpighian tubules; fb, fat body; rec, rectum, spm, spermatheca.
organs with antibodies against PER and TIM followed by appropriate fluorescent
markers allowed us to establish temporal patterns of the two clock proteins. We observed
that both PER and TIM were clearly rhythmic in several tissues in LD and DD. PER and
TIM peaked in the cell nuclei late at night, at ZT 22 (Fig. 2) and were at a minimum late
during the day, at ZT 8, essentially confirming the oscillatory pattern observed in
Malpighian tubules (Fig. 1). Entrained and free-running rhythms of PER and TIM were
detected in most segments of the alimentary canal, including esophagus, crop,
proventriculus (a.k.a. cardia), hindgut, and rectum. PER and TIM were also rhythmic in
all examined regions of the fat body, including the subcutaneous fat layer, and fat cells
associated with the gut and the reproductive organs. More discrete staining patterns were
observed in the female reproductive system. Rhythmic and nuclear expression of PER
and TIM were evident in the paired spermathecae and paraovaria, but the signal was
absent in the seminal receptacle, the lateral and common oviducts, and uterus. Similarly
discrete distribution of clock proteins was observed in the male reproductive system:
oscillations in PER and TIM were detected in the testis base, seminal vesicles, and
ejaculatory ducts, but not in the main body of the testes or paragonial accessory glands
(B. Gvakharia, personal communication). Finally, some tissues including the epidermis,
skeletal muscles and tracheal epithelium did not show detectable levels of either PER or

35
TIM, demonstrating that clock proteins are not ubiquitously present in all tissues but
rather are limited to specific, albeit multiple, organs or their specific segments.
A few studies performed on other insects provide additional evidence of clock
genes being active in peripheral tissues. Daily oscillations ofper mRNA and protein were
detected in the larval gut of the silkworm, Antheraea pernyi (Sauman and Reppert 1998),
and in the reproductive system of the codling moth, Cydia pomonella (Gvakharia et al.
2000). Insects are not an exception with regard to widespread activity of the timing genes
in their bodies; a similar picture has emerged from studies of vertebrates. From fishes to
mammals, mRNAs coding for elk, BMALl (the vertebrate equivalent of deye), and per
were detected in organs such as heart, lungs, kidney, and testis (King et al. 1997; Tei et
al. 1997; Whitmore et al. 1998; Yamazaki et al. 2000).
3. AUTONOMY OF THE PERIPHERAL CLOCKS.
The cycling of clock proteins in different fly tissues shows remarkable synchrony
of phase (with the exception of certain brain neurons (see, Kaneko 1998). The times of
nuclear translocation of PER and TIM and the times of their maximal expression are
similar in peripheral organs (Fig. 1 and 2), in the brains of adult flies (Stanewsky et al.
1997), and in the lateral neurons of larval and pupal brains (Kaneko et al. 1997; 2000;
Ivanchenko, submitted). There are two possible explanations for the synchrony between
peripheral oscillators and those located in the brain. First, the brain oscillator entrained
by the LD cycles could coordinate peripheral oscillators via blood-borne factor(s).
Second, central and peripheral oscillators could operate independently, achieving
synchronization via direct entrainment to the LD cycles. Several lines of experimental
evidence, discussed below, support the second possibility.
One piece of early evidence for the existence of autonomous circadian clocks in
insects came from studying the rhythms of sperm release in moths. In many moth
species, the release of sperm bundles from the testis to the vas deferens shows a daily
rhythm (reviewed by Giebultowicz 1999). The rhythm continues in cultured testis-vas
deferens complexes and can be entrained in vitro by LD cycles (Giebultowicz et al.
1989). Thus, a whole circadian system including photoreceptors, the clock pacemaker
and the output rhythms, is located in this specific portion of the male moth reproductive
system. Importantly, the very same tissues rhythmically express per mRNA and PER
protein (Gvakharia et al. 2000). Another example of a brain-independent cycle is the
daily pattern of cuticle deposition in the integument of some insects, which arises due to
rhythmic changes in the orientation of secreted cuticular layers (Neville 1970). This
rhythmic activity continues in pieces of integument cultured in vitro, providing indirect
evidence for the existence of an autonomous circadian oscillator in epidermal cells
(Weber 1995)
Convincing evidence for the existence of autonomous local oscillators was gained
using transgenic D. melanogaster that express luciferase under per or tim regulatory
sequences {per-hxc and tim-hxc lines); luciferase acts as a real time reporter for the
activity of the respective clock genes (Brandes et al. 1996). Owing to the fact that such
transgenic flies produce measurable light proportional to clock gene activities, one can
determine whether those genes continue to oscillate in internal organs cultured in vitro. A
group of tissues with self-sustained and light-entrainable rhythms of per was identified

36
this way. One example is the prothoracic (ring) gland, which produces the insects molting
hormone ecdysone. In fly pupae, per gene and protein are rhythmically expressed in the
ring gland and this expression continues in vitro (Emery et al. 1997). Rhythms inper-hic
activity were also found in chemosensory hairs located on the flies' antennae, proboscis,
wing margin, and legs maintained in vitro; these rhythms persisted in DD and were
shifted in response to a change in the LD cycle (Plautz et al. 1997). We recently
demonstrated that both per:MC and tim:uc oscillate in hindgut-rectum complexes and
Malpighian tubules in vitro (Giebultowicz et al. 2000). These oscillations persisted in
constant darkness with a period of nearly 24 h, and oscillation amplitude increased upon
return to LD, consistent with the sensitivity of these tissues to changes in the
environmental LD cycles.
The assumption that different local time-keeping centers widely distributed in the
fly body may be autonomous in their entrainment to the environmental LD cycles
requires that these clocks are equipped with their own light-sensing devices. Some organs
in Drosophila are not innervated, thus lacking any direct connection with the fly external
photoreceptors. We have recently studied the circadian photoreception in such an organ,
the Malpighian tubules. We determined that the blue light circadian photoreceptor
CRYPTOCHROME (CRY), recently identified in plants and insects (reviewed by Hall
2000), is present in Malpighian tubules. This photoreceptor entrains the clock in
Malpighian tubules, as it does the central clock in the brain (Stanewsky et al. 1998). CRY
mediates rapid degradation of the clock protein TIM, known to entrain the fly clock in
response to the environmental dark-light changes (Young 1998) both in Malpighian
tubules and in the brain lateral neurons. In addition to CRY, the clock in Malpighian
tubules is entrained by an unknown tissue-autonomous mechanism that does not directly
affect the level of TIM (Ivanchenko et al, submitted).
The physiological and molecular evidence presented above support the hypothesis
that the peripheral oscillators in insects are self-sustained and photoreceptive when
cultured in vitro. One may expect that these oscillators would also have a high degree of
autonomy in vivo, although phase-imposing effects of the brain clock cannot be excluded
a priori. To address this issue we monitored the free-running cycles of TIM protein in
Drosophila Malpighian tubules that were transplanted to host flies entrained to reverse
LD cycles with respect to the donor flies. TIM in the transplanted tubules cycled 12 hours
out of phase compared to host tubules, suggesting that different clocks in one organism
may operate independently despite sharing the same hormonal milieu (Giebultowicz et al.
2000). It appears, based on these observations, that circadian coordination of
physiological sub-systems in insects may be achieved via direct entrainment of light-
sensitive autonomous oscillators by enviromnental signals.
4. ACTIVATION OF CLOCK GENES DURING METAMORPHOSIS.
One of the important considerations in trying to understand how the insect
circadian system is organized relates to the developmental origins of the circadian clocks.
It has been known that the phase of adult behavioral rhythms in fruit flies can be set by a
pulse of light applied in early larval life to insects otherwise held in DD (reviewed in
(Kaneko 1998). Examination of PER and TIM staining patterns in the CNS revealed that
their cyclic expression persist in the lateral neurons of larval and pupal brains, providing

37
1 P+48 P+72 P+96 E E+24 1
Figure 3. Activation of tim and per genes during metamorphosis in selected tissues of D.
melanogaster, as shown by the by per-LsicZ and tim-G¥V reporters. The levels of
expression were scored subjectively as negative (white), intermediate (gray) and strong
(black). Developmental stages (see text) are indicated at the bottom. Tissues from 3
independently transformed lines for each reporter gave similar results. No fluorescence
was observed in control flies carrying the GFP reporter without the tim regulatory
regions.
the molecular basis for circadian time memory (Kaneko et al. 1997). Unlike the lateral
neurons in the brain, which are preserved from larvae to adult, most adult internal organs
differentiate de novo from the larval histoblast during metamorphosis. We examined the
developmental activation of the two clock gQnQS,per and tim, in selected fly organs using
transgenic flies carrying per-lacZ (Stanewsky et al. 1997) or tim-G¥? (Kaneko and Hall
2000) reporter constructs. Developing adults were staged according to Bainbridge and
Bownes (1981) and examined with respect to expression of clock genes at approximately
24 h intervals after pupariation. Activity of lacZ was detected in fixed organs as
described (Hege et al. 1997), and the intensity of GFP signal was observed in live organs
using a Zeiss Axiovert microscope with a GFP-optimized filter set. In all examined
organs, the onset of both per and tim gene expression occurred simultaneously; this may
signify the activation of local clock fiinction, since both genes are required for initiation
of circadian cycles. However, the activation of clock genes occurred at different
metamorphic stages in different organs (Fig. 3), indicating that it is developmentally
regulated, but in a tissue-specific way. The earliest expression of per and tim was
detected approximately 48 hours after puparium formation in the four rectal pads but not
in other parts of the rectum (Fig. 4). Activation of the genes in the male seminal vesicles
occurred approximately 24 hours later (P+72). Several other tissues, including the
hindgut and the remainder of the rectum, showed weak expression of clock genes one day
before adult eclosion (P+96) and a strong expression 24 h later, at the time of eclosion.
Finally, the Malpighian tubules were the last tissue in which the timing mechanism was
activated; weak expression of per and tim was detected at eclosion and maximal
expression was achieved one day after eclosion.
To gain a more complete picture of the developmental pattern of clock gene
expression, we also studied the activity of such genes in larvae ofD. melanogaster. Most
larval tissues die during metamorphosis and adult tissues differentiate de novo within the

38
tim-GFF perAacZ
Figure 4. Expression of tim as reported by //>w-GFP andper as reported hyper-lacZ in the
rectum of D. melanogaster 72 hours after pupariation. The four rectal pads are strongly
expressing both genes at this time. Note the excretory material in the rectum appearing
like granulated deposits on the/^er-lacZ image.
pupal case, with the exception of the Malpighian tubules, which ftinction as an excretory
organ in larvae and then survive metamorphosis to resume the same ftinction in adults.
Do larval tubules harbor a circadian oscillator? The answer appears to be negative, since
immunocytochemistry failed to detect PER or TIM proteins in larval tubules and tim-
GFP reporter gave no signal (Giebultowicz, unpublished). Thus, this tissue presents an
interesting case where the timing mechanism is absent in the organ at the larval stage but
becomes active in the same organ in adults. We also tested the digestive and excretory
systems taken from third instar larvae carrying tim-GW and did not detect tim activity in
these tissues. The widespread presence of the timing oscillators in the alimentary tract of
adults but not in larvae may be related to different lifestyles and feeding habits of the two
life stages. Larvae seem to feed continually, whereas adults may feed periodically in
correlation with their rest/activity cycles, although this was not yet rigorously tested.
The recruitment of timing mechanisms in specific physiological contexts is
supported by the fact that the expression of clock genes is initiated at different times in
different internal organs during adult development. Such tissue-by-tissue activation of
clock genes suggests that peripheral clocks may be turned on by tissue-specific signals,
rather than by a central mechanism. The independent onset of clock gene expression is
likely to be correlated with tissue-specific needs for circadian synchronization. The very
early activation of clock genes in the developing adult rectal pads may be related to the
accumulation of excretory material observed in the rectum during metamorphosis (Fig 4).
One may speculate that a putative oscillator in the rectal epithelium may be required for
excretory cycles associated with metamorphic development.
5. FUNCTIONS OF THE INSECT PERIPHERAL OSCILLATORS.
The oscillations of clock molecules in insect peripheral organs lead to the
expectation that these local oscillators impose daily rhythms on tissue-specific processes.

39
Unfortunately, cellular and physiological outputs are not yet known for most of the newly
identified peripheral oscillators, therefore, their clock status must remain tentative.
However, there are few cases in which the role of local oscillators has become apparent,
providing the first evidence that these oscillators are essential components of the multi-
oscillatory circadian system that seems to operate in insects.
One prominent example of a biologically relevant local oscillator is the brain-
independent clock located in the vas deferens of male moths. Several circadian rhythms
occurring in daily succession were identified in this complex (reviewed by Giebultowicz
1999). First, sperm is released from the testis during a circadian gate at the end of the day
and stored in the vas deferens. This rhythm is correlated with the rhythmic secretion of
glycoproteins from vas deferens epithelium into its lumen. Several hours later, sperm is
pushed out of the vas deferens by daily increases in contraction intensity of the vas
deferens wall. Thus, a locally operating clock appears to synchronize multiple rhythms
involved in sperm release and maturation. Disruption of the circadian rhythms by
constant light leads to male sterility, demonstrating their critical role in reproduction. A
similar mechanism may operate in D. melanogaster. A putative circadian oscillator was
identified in specific parts of the male fly reproductive system that are involved in sperm
release and maturation (Gvakharia et al, in preparation). Involvement of local circadian
clocks in fly reproduction is further suggested by the fact that the activation of clock
genes in the male reproductive tissues (Fig. 3) precedes the first sperm release in
developing adults (Giebultowicz, unpublished).
A defined output function has also been assigned to the peripheral oscillators in
the chemosensory hairs on the fly antennae. These organs display rhythm in
electrophysiological responses to two different classes of olfactory stimuli. Genetic tests
demonstrated that olfactory rhythms are driven by the oscillations of locally expressed
clock genes, rather than those active in the central brain (Krishnan et al. 1999).
Although the biological significance of the multiple insect peripheral clocks
remains obscure, physiologicalfimctionsmay emerge from co-localization of clock genes
with rhythmic output genes. A case in point is the identification of oscillatory expression
of the takeout gene, which codes for a ligand-binding protein, in segments of the
alimentary canal (Sarov-Blat et al. 2000) in which we observed cycling of PER and TIM
proteins.
6. CONCLUSIONS
The existence of multi-oscillatory systems in complex animals was predicted in
the past based on the dissimilar characteristics of output rhythms in single organisms
(see, for example, Saunders 1986; Tossini and Menaker 1998). The use of molecular
tools for mapping the activity of clock genes confirms these predictions by revealing the
widespread distribution of these genes in peripheral organs. Rhythmic expression of
clock genes that free-runs in constant darkness provide the initial clue as to which insect
organs may posses circadian clock function. However, final proof of such function will
be the identification of physiological output rhythms. This has not been achieved for most
clock-positive tissues. An even bigger future task is developing an understanding of the
regulatory cascades leading from the clock genes to overt rhythms via clock-controlled
effector molecules.

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CHILDREN IN PRISON
AND
OTHER CRUELTIES
OF
PRISON LIFE.
MURDOCH & CO.,
26, Paternoster Square,
London.

PUBLISHERS' NOTE.
The circumstance which called forth this letter is a woeful one for
Christian England. Martin, the Reading warder, is found guilty of
feeding the hungry, nursing the sick, of being kindly and humane.
These are his offences in plain unofficial language.
This pamphlet is tendered to earnest persons as evidence that the
prison system is opposed to all that is kind and helpful. Herein is
shown a process that is dehumanizing, not only to the prisoners, but
to every one connected with it.
Martin was dismissed. It happened in May last year. He is still out of
employment and in poor circumstances. Can anyone help him?
February, 1898.

SOME CRUELTIES OF PRISON LIFE.
THE EDITOR OF THE DAILY CHRONICLE.
Sir,—I learn with great regret, through an extract from the columns
of your paper, that the warder Martin, of Reading Prison, has been
dismissed by the Prison Commissioners for having given some sweet
biscuits to a little hungry child. I saw the three children myself on
the Monday preceding my release. They had just been convicted,
and were standing in a row in the central hall in their prison dress,
carrying their sheets under the arms previous to their being sent to
the cells allotted to them. I happened to be passing along one of the
galleries on my way to the reception room, where I was to have an
interview with a friend. They were quite small children, the youngest
—the one to whom the warder gave the biscuits—being a tiny little
chap, for whom they had evidently been unable to find clothes small
enough to fit. I had, of course, seen many children in prison during
the two years during which I was myself confined. Wandsworth
Prison, especially, contained always a large number of children. But
the little child I saw on the afternoon of Monday, the 17th, at
Reading, was tinier than any one of them. I need not say how
utterly distressed I was to see these children at Reading, for I knew
the treatment in store for them. The cruelty that is practised by day
and night on children in English prisons is incredible, except to those
who have witnessed it and are aware of the brutality of the system.
People nowadays do not understand what cruelty is. They regard it
as a sort of terrible mediæval passion, and connect it with the race
of men like Eccelin da Romano, and others, to whom the deliberate
infliction of pain gave a real madness of pleasure. But men of the
stamp of Eccelin are merely abnormal types of perverted
individualism. Ordinary cruelty is simply stupidity. It comes from the
entire want of imagination. It is the result in our days of stereotyped

systems, of hard-and-fast rules, of centralisation, of officialism, and
of irresponsible authority. Wherever there is centralisation there is
stupidity. What is inhuman in modern life is officialism. Authority is
as destructive to those who exercise it as it is to those on whom it is
exercised. It is the Prison Board, with the system that it carries out,
that is the primary source of the cruelty that is exercised on a child
in prison. The people who uphold the system have excellent
intentions. Those who carry it out are humane in intention also.
Responsibility is shifted on to the disciplinary regulations. It is
supposed that because a thing is the rule it is right.
The present treatment of children is terrible, primarily from people
not understanding the peculiar psychology of a child's nature. A child
can understand a punishment inflicted by an individual, such as a
parent or guardian, and bear it with a certain amount of
acquiescence. What it cannot understand is a punishment inflicted
by Society. It cannot realise what Society is. With grown people it is,
of course, the reverse. Those of us who are either in prison or have
been sent there, can understand, and do understand, what that
collective force called Society means, and whatever we may think of
its methods or claims, we can force ourselves to accept it.
Punishment inflicted on us by an individual, on the other hand, is a
thing that no grown person endures or is expected to endure.
The child consequently, being taken away from its parents by people
whom it has never seen, and of whom it knows nothing, and finding
itself in a lonely and unfamiliar cell, waited on by strange faces, and
ordered about and punished by the representatives of a system that
it cannot understand, becomes an immediate prey to the first and
most prominent emotion produced by modern prison life—the
emotion of terror. The terror of a child in prison is quite limitless. I
remember once in Reading, as I was going out to exercise, seeing in
the dimly-lit cell, right opposite my own, a small boy. Two warders,
not unkindly men, were talking to him, with some sternness
apparently, or perhaps giving him some useful advice about his
conduct. One was in the cell with him, the other was standing

outside. The child's face was like a white wedge of sheer terror.
There was in his eyes the mute appeal of a hunted animal. The next
morning I heard him at breakfast-time crying, and calling to be let
out. His cry was for his parents. From time to time I could hear the
deep voice of the warder on duty warning him to keep quiet. Yet he
was not even convicted of whatever little offence he had been
charged with. He was simply on remand. That I knew by his wearing
his own clothes, which seemed neat enough. He was, however,
wearing prison socks and shoes. This showed that he was a very
poor boy, whose own shoes, if he had any, were in a bad state.
Justices and magistrates, an entirely ignorant class as a rule, often
remand children for a week, and then perhaps remit whatever
sentence they are entitled to pass. They call this "not sending a child
to prison." It is, of course, a stupid view on their part. To a little
child, whether he is in prison on remand or after conviction, is a
subtlety of social position he cannot comprehend. To him the
horrible thing is to be there at all. In the eyes of humanity it should
be a horrible thing for him to be there at all.
This terror that seizes and dominates the child, as it seizes the
grown man also, is of course intensified beyond power of expression
by the solitary cellular system of our prisons. Every child is confined
to its cell for twenty-three hours out of the twenty-four. This is the
appalling thing. To shut up a child in a dimly-lit cell for twenty-three
hours out of the twenty-four is an example of the cruelty of stupidity.
If an individual, parent or guardian, did this to a child he would be
severely punished. The Society for the Prevention of Cruelty to
Children would take the matter up at once. There would be on all
hands the utmost detestation of whomsoever had been guilty of
such cruelty. A heavy sentence would undoubtedly follow conviction.
But our own actual society does worse itself, and to the child to be
so treated by a strange abstract force, of whose claims it has no
cognizance, is much worse than it would be to receive the same
treatment from its father or mother, or someone it knew. The
inhuman treatment of a child is always inhuman, by whomsoever it
is inflicted. But inhuman treatment by Society is to the child the

more terrible because there is no appeal. A parent or guardian can
be moved, and let out the child from the dark lonely room in which it
is confined. But a warder cannot. Most warders are very fond of
children. But the system prohibits them from rendering the child any
assistance. Should they do so, as Warder Martin did, they are
dismissed.
The second thing from which a child suffers in prison is hunger. The
food that is given to it consists of a piece of usually badly-baked
prison bread and a tin of water for breakfast at half-past seven. At
twelve o'clock it gets dinner, composed of a tin of coarse Indian meal
stirabout, and at half-past five it gets a piece of dry bread and a tin
of water for its supper. This diet in the case of a strong grown man
is always productive of illness of some kind, chiefly of course
diarrhœa, with its attendant weakness. In fact in a big prison
astringent medicines are served out regularly by the warders as a
matter of course. In the case of a child, the child is, as a rule,
incapable of eating the food at all. Anyone who knows anything
about children knows how easily a child's digestion is upset by a fit
of crying, or trouble and mental distress of any kind. A child who has
been crying all day long, and perhaps half the night, in a lonely
dimly-lit cell, and is preyed upon by terror, simply cannot eat food of
this coarse, horrible kind. In the case of the little child to whom
Warder Martin gave the biscuits, the child was crying with hunger on
Tuesday morning, and utterly unable to eat the bread and water
served to it for its breakfast. Martin went out after the breakfasts
had been served and bought the few sweet biscuits for the child
rather than see it starving. It was a beautiful action on his part, and
was so recognised by the child, who, utterly unconscious of the
regulation of the Prison Board, told one of the senior warders how
kind this junior warder had been to him. The result was, of course, a
report and a dismissal.
I know Martin extremely well, and I was under his charge for the
last seven weeks of my imprisonment. On his appointment at
Reading he had charge of Gallery C, in which I was confined, so I

saw him constantly. I was struck by the singular kindness and
humanity of the way in which he spoke to me and to the other
prisoners. Kind words are much in prison, and a pleasant "Good
morning" or "Good evening" will make one as happy as one can be
in solitary confinement. He was always gentle and considerate. I
happen to know another case in which he showed great kindness to
one of the prisoners, and I have no hesitation in mentioning it. One
of the most horrible things in prison is the badness of the sanitary
arrangements. No prisoner is allowed under any circumstances to
leave his cell after half-past five p.m. If, consequently, he is suffering
from diarrhœa, he has to use his cell as a latrine, and pass the night
in a most fetid and unwholesome atmosphere. Some days before my
release Martin was going the rounds at half-past seven with one of
the senior warders for the purpose of collecting the oakum and tools
of the prisoners. A man just convicted, and suffering from violent
diarrhœa in consequence of the food, as is always the case, asked
this senior warder to allow him to empty the slops in his cell on
account of the horrible odour of the cell and the possibility of illness
again in the night. The senior warder refused absolutely; it was
against the rules. The man, as far as he was concerned, had to pass
the night in this dreadful condition. Martin, however, rather than see
this wretched man in such a loathsome predicament, said he would
empty the man's slops himself, and did so. A warder emptying a
prisoner's slops is, of course, against the rules, but Martin did this
act of kindness to the man out of the simple humanity of his nature,
and the man was naturally most grateful.
As regards the children, a great deal has been talked and written
lately about the contaminating influence of prison on young children.
What is said is quite true. A child is utterly contaminated by prison
life. But the contaminating influence is not that of the prisoners. It is
that of the whole prison system—of the governor, the chaplain, the
warders, the lonely cell, the isolation, the revolting food, the rules of
the Prison Commissioners, the mode of discipline as it is termed, of
the life. Every care is taken to isolate a child from the sight even of
all prisoners over sixteen years of age. Children sit behind a curtain

in chapel, and are sent to take exercise in small sunless yards—
sometimes a stone-yard, sometimes a yard at the back of the mills—
rather than that they should see the elder prisoners at exercise. But
the only really humanising influence in prison is the influence of the
prisoners. Their cheerfulness under terrible circumstances, their
sympathy for each other, their humility, their gentleness, their
pleasant smiles of greeting when they meet each other, their
complete acquiescence in their punishments, are all quite wonderful,
and I myself learnt many sound lessons from them. I am not
proposing that the children should not sit behind a curtain in chapel,
or that they should take exercise in a corner of the common yard. I
am merely pointing out that the bad influence on children is not, and
could never be, that of the prisoners, but is, and will always remain,
that of the prison system itself. There is not a single man in Reading
Gaol that would not gladly have done the three children's
punishment for them. When I saw them last it was on the Tuesday
following their conviction. I was taking exercise at half-past eleven
with about twelve other men, as the three children passed near us,
in charge of a warder, from the damp, dreary stone-yard in which
they had been at their exercise. I saw the greatest pity and
sympathy in the eyes of my companions as they looked at them.
Prisoners are, as a class, extremely kind and sympathetic to each
other. Suffering and the community of suffering makes people kind,
and day after day as I tramped the yard I used to feel with pleasure
and comfort what Carlyle calls somewhere "the silent rhythmic
charm of human companionship." In this as in all other things,
philanthropists and people of that kind are astray. It is not the
prisoners who need reformation. It is the prisons.
Of course no child under fourteen years of age should be sent to
prison at all. It is an absurdity, and, like many absurdities, of
absolutely tragic results. If, however, they are to be sent to prison,
during the daytime they should be in a workshop or schoolroom with
a warder. At night they should sleep in a dormitory, with a night-
warder to look after them. They should be allowed exercise for at
least three hours a day. The dark, badly-ventilated, ill-smelling prison

cells are dreadful for a child, dreadful indeed for anyone. One is
always breathing bad air in prison. The food given to children should
consist of tea and bread-and-butter and soup. Prison soup is very
good and wholesome. A resolution of the House of Commons could
settle the treatment of children in half an hour. I hope you will use
your influence to have this done. The way that children are treated
at present is really an outrage on humanity and common-sense. It
comes from stupidity.
Let me draw attention now to another terrible thing that goes on in
English prisons, indeed in prisons all over the world where the
system of silence and cellular confinement is practised. I refer to the
large number of men who become insane or weak-minded in prison.
In convict prisons this is, of course, quite common; but in ordinary
gaols also, such as that I was confined in, it is to be found.
About three months ago, I noticed amongst the prisoners who took
exercise with me a young man who seemed to me to be silly or half-
witted. Every prison of course has its half-witted clients, who return
again and again, and may be said to live in the prison. But this
young man struck me as being more than usually half-witted on
account of his silly grin and idiotic laughter to himself, and the
peculiar restlessness of his eternally twitching hands. He was noticed
by all the other prisoners on account of the strangeness of his
conduct. From time to time he did not appear at exercise, which
showed me that he was being punished by confinement to his cell.
Finally, I discovered that he was under observation, and being
watched night and day by warders. When he did appear at exercise,
he always seemed hysterical, and used to walk round crying or
laughing. At chapel he had to sit right under the observation of two
warders, who carefully watched him all the time. Sometimes he
would bury his head in his hands, an offence against the chapel
regulations, and his head would be immediately struck up by a
warder, so that he should keep his eyes fixed permanently in the
direction of the Communion-table. Sometimes he would cry—not
making any disturbance—but with tears streaming down his face

and a hysterical throbbing in the throat. Sometimes he would grin
idiot-like to himself and make faces. He was on more than one
occasion sent out of chapel to his cell, and of course he was
continually punished. As the bench on which I used to sit in chapel
was directly behind the bench at the end of which this unfortunate
man was placed, I had full opportunity of observing him. I also saw
him, of course, at exercise continually, and I saw that he was
becoming insane, and was being treated as if he was shamming.
On Saturday week last, I was in my cell at about one o'clock
occupied in cleaning and polishing the tins I had been using for
dinner. Suddenly I was startled by the prison silence being broken by
the most horrible and revolting shrieks or rather howls, for at first I
thought some animal like a bull or a cow was being unskilfully
slaughtered outside the prison walls. I soon realised, however, that
the howls proceeded from the basement of the prison, and I knew
that some wretched man was being flogged. I need not say how
hideous and terrible it was for me, and I began to wonder who it
was who was being punished in this revolting manner. Suddenly it
dawned upon me that they might be flogging this unfortunate
lunatic. My feelings on the subject need not be chronicled; they have
nothing to do with the question.
The next day, Sunday 16th, I saw the poor fellow at exercise, his
weak, ugly, wretched face bloated by tears and hysteria almost
beyond recognition. He walked in the centre ring along with the old
men, the beggars and the lame people, so that I was able to
observe him the whole time. It was my last Sunday in prison, a
perfectly lovely day, the finest day we had had the whole year, and
there, in the beautiful sunlight, walked this poor creature—made
once in the image of God—grinning like an ape, and making with his
hands the most fantastic gestures, as though he was playing in the
air on some invisible stringed instrument, or arranging and dealing
counters in some curious game. All the while these hysterical tears,
without which none of us ever saw him, were making soiled runnels
on his white swollen face. The hideous and deliberate grace of his

gestures made him like an antic. He was a living grotesque. The
other prisoners all watched him, and not one of them smiled.
Everybody knew what had happened to him, and that he was being
driven insane—was insane already. After half-an-hour, he was
ordered in by the warder, and, I suppose, punished. At least he was
not at exercise on Monday, though I think I caught sight of him at
the corner of the stone-yard, walking in charge of a warder.
On the Tuesday—my last day in prison—I saw him at exercise. He
was worse than before, and again was sent in. Since then I know
nothing of him, but I found out from one of the prisoners who
walked with me at exercise that he had had twenty-four lashes in
the cook-house on Saturday afternoon, by order of the visiting
justices on the report of the doctor. The howls that had horrified us
all were his.
This man is undoubtedly becoming insane. Prison doctors have no
knowledge of mental disease of any kind. They are as a class
ignorant men. The pathology of the mind is unknown to them. When
a man grows insane, they treat him as shamming. They have him
punished again and again. Naturally the man becomes worse. When
ordinary punishments are exhausted, the doctor reports the case to
the justices. The result is flogging. Of course the flogging is not
done with a cat-of-nine-tails. It is what is called birching. The
instrument is a rod; but the result on the wretched half-witted man
may be imagined.
His number is, or was, A. 2. 11. I also managed to find out his
name. It is Prince. Something should be done at once for him. He is
a soldier, and his sentence is one of court-martial. The term is six
months. Three have yet to run.
May I ask you to use your influence to have this case examined into,
and to see that the lunatic prisoner is properly treated?
No report by the Medical Commissioners is of any avail. It is not to
be trusted. The medical inspectors do not seem to understand the

difference between idiocy and lunacy—between the entire absence
of a function or organ and the diseases of a function or organ. This
man A. 2. 11, will, I have no doubt, be able to tell his name, the
nature of his offence, the day of the month, the date of the
beginning and expiration of his sentence, and answer any ordinary
simple question; but that his mind is diseased admits of no doubt. At
present it is a horrible duel between himself and the doctor. The
doctor is fighting for a theory. The man is fighting for his life. I am
anxious that the man should win. But let the whole case be
examined into by experts who understand brain-disease, and by
people of humane feelings who have still some common-sense and
some pity. There is no reason that the sentimentalist should be
asked to interfere. He always does harm. He culminates at his
starting point. His end, as his origin, is an emotion.
The case is a special instance of the cruelty inseparable from a
stupid system, for the present Governor of Reading is a man of
gentle and humane character, greatly liked and respected by all the
prisoners. He was appointed in July last, and though he cannot alter
the rules of the prison system, he has altered the spirit in which they
used to be carried out under his predecessor. He is very popular with
the prisoners and with the warders. Indeed he has quite elevated
the whole tone of the prison-life. Upon the other hand, the system is
of course beyond his reach as far as altering its rules is concerned. I
have no doubt that he sees daily much of what he knows to be
unjust, stupid, and cruel. But his hands are tied. Of course I have no
knowledge of his real views of the case of A. 2. 11, nor, indeed, of
any of his views on our present system. I merely judge him by the
complete change he brought about in Reading Prison. Under his
predecessor the system was carried out with the greatest harshness
and stupidity.—I remain, Sir, your obedient servant,
OSCAR WILDE.
France, May 27th, 1897.

Transcriber's notes:
The following is a list of changes made to the original. The first line is the original line,
the second the corrected one.
whom Warder Martin gave the buscuits, the child was
whom Warder Martin gave the biscuits, the child was
sight of him at the corner of the stoneyard, walking in
sight of him at the corner of the stone-yard, walking in

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