Integrative and Comparative Biology
Integrative and Comparative Biology, volume 53, number 1, pp. 131–143
doi:10.1093/icb/ict023
Society for Integrative and Comparative Biology
SYMPOSIUM
Evolutionary Links Between Circadian Clocks and Photoperiodic
Diapause in Insects
Megan E. Meuti1 and David L. Denlinger
Department of Entomology, The Ohio State University, Columbus, OH 43210, USA
From the symposium ‘‘Keeping Time During Animal Evolution: Conservation and Innovation of the Circadian Clock’’
presented at the annual meeting of the Society for Integrative and Comparative Biology, January 3–7, 2013 at San
Francisco, California.
1
E-mail: [email protected]
Synopsis In this article, we explore links between circadian clocks and the clock involved in photoperiodic regulation of
diapause in insects. Classical resonance (Nanda–Hamner) and night interruption (Bünsow) experiments suggest a circadian basis for the diapause response in nearly all insects that have been studied. Neuroanatomical studies reveal physical
connections between circadian clock cells and centers controlling the photoperiodic diapause response, and both mutations and knockdown of clock genes with RNA interference (RNAi) point to a connection between the clock genes and
photoperiodic induction of diapause. We discuss the challenges of determining whether the clock, as a functioning
module, or individual clock genes acting pleiotropically are responsible for the photoperiodic regulation of diapause,
and how a stable, central circadian clock could be linked to plastic photoperiodic responses without compromising the
clock’s essential functions. Although we still lack an understanding of the exact mechanisms whereby insects measure day/
night length, continued classical and neuroanatomical approaches, as well as forward and reverse genetic experiments, are
highly complementary and should enable us to decipher the diverse ways in which circadian clocks have been involved in
the evolution of photoperiodic induction of diapause in insects. The components of circadian clocks vary among insect
species, and diapause appears to have evolved independently numerous times, thus, we anticipate that not all photoperiodic clocks of insects will interact with circadian clocks in the same fashion.
Introduction
The daily rotation of the earth on its axis and the
annual revolution of the earth around the sun generate patterns that have profound impacts on daily
and seasonal activity patterns of organisms. Feeding,
mating, sleeping, and indeed most behavioral and
physiological activities show a distinct rhythm with
a peak of activity occurring at a certain time of the
daily light:dark cycle. A circadian rhythm is defined
as one that persists with a periodicity of approximately 24 h, even after the organism is shifted from
a light:dark cycle to constant darkness. This feature
suggests that the rhythm is endogenous rather than
an immediate response to the external light:dark
cycle. The other major rhythm is based on the seasonal calendar known as photoperiodism. In this
case, the organism responds either to annual changes
in day length, or directly to the length of day/night,
and uses this information to determine the timing of
key events such as migration or the entry into a
dormant state. Both circadian and photoperiodic
events rely on an ability to precisely measure time.
In this article, we discuss evolutionary links between
these two timekeeping mechanisms with a special
focus on photoperiodic induction of diapause in
insects.
Circadian clocks
Circadian clocks correctly time a range of important
behavioral and physiological processes in animals
including sleep–wake cycles (Czeisler et al. 1986),
locomotion patterns (Silver et al. 1996), feeding
(Nishio et al. 1979), mating (Sakai and Ishida
2001), and cell division (Matsuo et al. 2003). Given
Advanced Access publication April 24, 2013
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132
the adaptive importance of circadian clocks (Sharma
2003), it is not surprising such clocks have been
documented frequently in organisms ranging from
photosensitive cyanobacteria to humans. The key
genes involved in animal circadian clocks are highly
conserved. For example, there is high homology
among core insect and human circadian clock
genes, and recent work on circadian clocks in the
cnidarian, Nematostella vectensis (Reitzel et al.
2010), reveals a high similarity of the cnidarian
clock to both insect and mammalian clocks, thus
implying an early evolution of clocks within animals.
Konopka and Benzer (1971) first identified the
period gene in Drosophila melanogaster after screening
for mutants that were arrhythmic and had altered
circadian periods. Since then, additional components
of circadian clocks in insects have been identified,
and we now have a mechanistic understanding of
circadian function in insects. In the circadian clock
model for Drosophila (Fig. 1A), the proteins CLOCK
(CLK) and CYCLE (CYC) act as positive transcriptional regulators by binding to an E-box promoter
on the genes period (per) and timeless (tim) and
recruit RNA polymerase, thereby increasing the
abundance of per and tim mRNAs. Once in the cytosol, per and tim mRNAs are translated into PER
and TIM proteins, which then form a heterodimer
that translocates back into the nucleus where the
PER component of the heterodimer acts to inhibit
action of CLK and CYC, thereby suppressing
transcription of itself and tim. Circadian clocks are
informed by light through the action of
CRYPTOCHROME1 (CRY1) which binds to TIM
in the presence of light and degrades both TIM
and itself.
For years, it was assumed that circadian clocks in
other insects functioned like clocks in Drosophila, but
the discovery that the monarch butterfly, Danaus
plexippus, has an additional clock protein
CRYPTOCHROME2 (CRY2; Zhu et al. 2005) suggested that this may not be the case. Unlike CRY1,
CRY2 is light insensitive and acts as a potent
repressor of CLK and CYC, much like the mammalian CRYs. Zhu et al. (2005) also identified
CRY2 in other Lepidoptera, as well as in the
mosquito Anopheles gambiae, and the red flour
beetle Tribolium castaneum. CRY2 has since been
found in every non-drosophilid insect examined
(Yuan et al. 2007), and hence our understanding
of circadian clocks in insects has been greatly
enhanced. We now know that CRY2, not PER, is
likely the important negative regulator of circadian
clocks in most, if not all, non-drosophilid insects
(Fig. 1B).
M. E. Meuti and D. L. Denlinger
Fig. 1 The circadian clock model in Drosophila and other insects.
(A) In the nucleus of D. melanogaster, circadian cells CLOCK
(CLK) and CYCLE (CYC) proteins form a heterodimer that acts
as a transcriptional activator by binding to the E-box promoter
region of the period (per) and timeless (tim) genes. per and tim
mRNA are translated in the cytoplasm of the cell. PER and TIM
proteins then form a heterodimer and translocate back into the
nucleus where PER inhibits the action of CLK:CYC, thereby
suppressing the transcription of per and tim. The
CRYPTOCHROME1 (CRY1) protein degrades TIM and itself in
the presence of light. This results in increasing levels of per and
tim mRNA throughout the day when CLK:CYC activity is uninhibited, and decreasing levels of per and tim mRNA during the
night. (B) The circadian clock in most other insects, such as
monarch butterflies, and mosquitoes, differ from the Drosophila
clock in that they possess a light-insensitive CRY2 protein
that acts as the major negative transcriptional regulator of the
core circadian clock. In this case, PER appears to assist CRY2
in nuclear translocation, whereas TIM helps to stabilize PER
and CRY2.
Photoperiodism, the seasonal clock
Just as the earth’s rotation about its axis produces a
daily pattern of light and dark, the earth’s 23.58 tilt
as it rotates about the sun produces an annual pattern of changing day length, temperature, and
Insect circadian clocks and photoperiodic diapause
precipitation. Consequently, some seasons are favorable for growth, development, and reproduction
while others are not. Seasonal timing is just as important as daily timing and is essential for enabling
animals to correctly anticipate seasonal environmental changes. In temperate and polar environments,
where seasonal differences are pronounced, many animals evolved the capacity to measure and respond
to changes in day length, or photoperiod. As the
same geographic location will experience the same
length of day or night on a specific day each year,
photoperiod is a much more reliable seasonal signal
than changes in temperature or rainfall, which vary
greatly over time. Photoperiod is the primary signal
that most temperate-zone animals, including insects,
use to anticipate seasonal change.
Among insects, one of the most prevalent photoperiodic responses is the onset of diapause. Diapause
is a programmed arrested state of development that
allows insects and other arthropods to survive adverse seasonal conditions either by becoming dormant locally, or by first migrating to a more
favorable environment. Tropical insects use a variety
of signals, such as changes in temperature, rainfall, or
population density, to initiate diapause, but the most
common signal that insects in temperate environments use is photoperiod (Tauber et al. 1986).
Although diverse responses to photoperiod have
been documented in temperate zones, most commonly short days (long nights) elicit a high incidence
of overwintering diapause, whereas long days (short
nights) evoke a low incidence of diapause (Fig. 2).
The point at which the incidence of diapause is 50%
of its maximal level is known as the critical photoperiod (CPP). The CPP among populations of the
same species increases with latitude and elevation
(Bradshaw and Lounibos 1977; Bradshaw and
Holzapfel 1975), thus enabling populations at
higher latitudes and those that reside at higher elevations to adjust to the earlier onset of winter by
entering diapause at an earlier date (i.e., increase
their CPP). The relationship between CPP and latitude/elevation demonstrates one of many characteristics of photoperiodic diapause that is plastic within
a species and is acted upon by natural selection.
Another defining characteristic of diapause in insects is that each species is typically capable of entering diapause in only a single life stage, although
diapause can occur in any of the life stages. For example, in the order Diptera, there are examples of
photoperiodically-induced embryonic diapause (the
Asian tiger mosquito, Aedes albopictus), larval diapause (the pitcher-plant mosquito, Wyeomyia
smithii), pupal diapause (the flesh fly, Sarcophaga
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Fig. 2 Photoperiodic response curve of diapausing pupae of the
flesh fly, Sarcophga bullata, from populations in Illinois and
Missouri. Each point represents the mean (SE) incidence of
diapause in progeny of 12–14 females (from Denlinger 1972).
bullata), and adult reproductive diapause (the northern house mosquito, Culex pipiens). Such differences
in stage-specificity of diapause within a single order,
and sometimes even within a single genus, suggest
that photoperiodic diapause has evolved multiple
times.
The ability to enter diapause has contributed to
the evolutionary success of insects (Denlinger 2008).
Insects in diapause are resistant to a range of environmental stresses, and these responses are mediated
through physiological mechanisms, such as the generation of polyols and heat shock proteins that enhance survival at low temperatures; elevation of
cuticular hydrocarbons that protect against desiccation; increased lipid stores and suppressed metabolic
rates that enable diapausing insects to survive long
periods without food; and boosts in immune responses to combat increased attacks by pathogens
(Denlinger 2002, 2008; Hahn and Denlinger 2011).
The hormonal regulators responsible for initiating
and maintaining diapause have also been well-characterized (Denlinger et al. 2012). A failure of the
brain and its associated endocrine organs to produce
the hormones that trigger molting (larval, nymphal,
and pupal diapauses) or maturation of reproductive
organs (adult reproductive diapause) is the dominant
endocrine mechanism that halts development.
Although we know photoperiod is the token stimulus that most temperate insects use to initiate their
overwintering diapause and we know a considerable
amount about the ultimate endocrine signals that
bring about this halt in development, we know
little about the mechanisms used by insects to
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measure day length and to translate this information
into the downstream endocrine signal.
Photoperiodic and circadian clock
models
Erwin Bünning (1936) hypothesized that plants and
animals likely use their circadian clocks to measure
day length, and hence initiate photoperiodic responses, because the circadian clock already provides
critical information on light/dark cycles. According
to Bünning’s hypothesis, also referred to as the external coincidence model, light entrains the circadian
clock which sets a light sensitive phase (’i) in the
late night/early morning, such that if ’i is illuminated, a long-day response is elicited. This model
has been tested extensively in evaluating several photoperiodic responses, including diapause (reviews by
Saunders 1981, 2009, 2010b, 2013; Saunders and
Bertossa 2011). One protocol for testing this
model, Bünsow (1953) or night interruption studies,
uses short photoperiods followed by brief pulses of
light administered at different times throughout an
extended night. If the light falls on ’i a long-day
response is elicited.
Nanda–Hamner resonance experiments have also
been used extensively; in this protocol, a short period
of light is followed by periods of darkness to create
light:dark cycles that range from 24 to 72 h (Nanda
and Hamner 1958). In such experiments, animals are
expected to show short-day responses when the total
period (T) of the light:dark cycle is equal to a multiple of 24 h, and a long-day response when T 6¼ n24
h (i.e., T ¼ 36). Over a range of T ’s, insects are expected to show peaks and troughs in the incidence of
diapause. Evidence from such experiments supports
the hypothesis that photoperiodic responses as diverse as diapause in flesh flies (Saunders 1973),
flowering in plants (Somers 2010), and reproduction
in Siberian hamsters (Prendergast et al. 2004) have a
circadian basis and can be explained using the external coincidence model.
Bradshaw et al. (2003a) demonstrated that there is
no relationship between changes in CPP and periodicity of peaks in the incidence of diapause in the
pitcher-plant mosquito W. smithii using Nanda–
Hamner experiments, but there was a negative correlation between CPP and the amplitude of these
peaks. To determine whether this correlation was
the result of a causal connection between circadian
oscillators and photoperiodic induction of diapause,
Bradshaw et al. (2012), using only five rounds of
antagonistic selection, were able to reverse their previously observed negative correlation between CPP
M. E. Meuti and D. L. Denlinger
and amplitude of diapause. They thus present this
as evidence that the circadian clock and photoperiodic responses can evolve independently. Although
we agree with their ultimate conclusion, their interpretation is predicated on the assumption that amplitude of the diapause response in the Nanda–
Hamner experiments has a circadian basis. This
may not be the case. Instead, the amplitude of the
diapause response may reflect light sensitivity of the
circadian oscillator (Tauber 2012), or may be a feature that has no connection to the circadian clock.
Although their selection experiments changed both
the CPP and amplitude of the diapause response,
there was no correlated change in the period between
peaks in the incidence of diapause. This indicates
that the underlying periodicity of the circadian pacemaker was not affected by their experiments.
Therefore, their ultimate conclusion that CPP, a
photoperiodic response, can be acted upon by natural selection independently of the clock is supported
when one considers the period between peaks, rather
than their amplitude, as critical in determining the
incidence of diapause. Mechanisms that could
achieve this dichotomy are discussed later in this
article.
A contrasting mechanism in which circadian
clocks could be involved in photoperiodic responses
is known as the internal coincidence model. In this
model, light entrains separate dawn and dusk oscillators, such that photoperiodic responses result from
changing phases between the two oscillators. This
model was first proposed by Pittendrigh and Minis
(1964) after it became apparent that most multicellular animals have multiple circadian oscillators that
may interact with each other in different ways.
Saunders (1974) used Nanda–Hamner experiments
to determine that the parasitic wasp, Nasonia vitripennis, possesses separate dawn and dusk oscillators,
and both oscillators are involved in programming the
wasp’s larval diapause.
It is also possible that circadian clocks are not
consistently involved in initiating photoperiodic responses. Instead, animals might measure day length
or night length through the accumulation of a chemical substance, such that a photoperiodic response is
initiated if the substance reaches a critical threshold.
Insects and other animals were believed to use such
an ‘‘hourglass timer’’ if they failed to show positive
responses to Bünsow or Nanda–Hamner experiments. Lees (1966, 1973) suggested that the vetch
aphid, Megoura viciae, uses an hourglass-like interval
timer to measure night length and initiate diapause,
although Vaz Nunes and Hardie (1993) later proposed that M. viciae measured extended periods of
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Insect circadian clocks and photoperiodic diapause
darkness repeatedly in a circadian-based manner. Vaz
Nunes and Veerman (1982) hypothesized that the
spider mite, Tetranychus urticae, which shows
strong positive responses to Nanda–Hamner experiments but a negative response to certain skeleton
photoperiods and T-experiments, uses an hourglass
clock to distinguish long from short nights, but a
circadian-based counter to tally the number of
long nights. Saunders (2010a) countered that the
observations on T. urticae could be explained by
the circadian-based external coincidence model by
considering the intensity and wavelengths of light
used in the T. urticae experiments.
Finally, Emerson et al. (2008) explored whether
the photoperiodic counter involved in terminating
diapause in the pitcher-plant mosquito, W. smithii,
is linked to the circadian clock. The authors did this
by exposing diapausing larvae from different populations to 18 h photophases followed by 8, 30, or 54 h
of darkness. They reasoned that if an internal coincidence model was responsible for breaking diapause,
fewer 8:54 cycles would be required to break diapause than if the mosquitoes were using an hourglass
or external coincidence counter. If, however, the
mosquitoes required the same number of light:dark
cycles to break diapause, this would imply that W.
smithii uses an hourglass or external coincidence
counter that measures total photoperiod. Among all
the populations they examined, the same number of
light:dark cycles were required to break diapause,
and hence the termination of diapause was considered to be a function of a photoperiodic counter that
measured day length. Although the results could not
distinguish between an external coincidence model
and an hourglass counter, they assumed that
W. smithii counted exogenous periods of environmental light, rather than internal circadian periods.
Saunders (2013) argues that a circadian basis for
counting photoperiods could also be inferred from
their experiments.
To date, nearly all classic experiments in plants,
vertebrates, and insects suggest a circadian basis for
photoperiodic responses. The hourglass model,
which assumes that a compound accumulates
throughout the day or night independently of circadian clocks, is appealing because of its inherent simplicity, but this model has not been widely supported
in studies on the photoperiodic induction of diapause. However, it remains quite possible that a photoperiodic counter, acting either with or downstream
of circadian clocks, could work by accumulating a
substance in the insect brain that, after reaching its
threshold, would initiate diapause. Insects are, of
course, incredibly diverse and there is no reason to
assume that all species use a single model to measure
photoperiodic time.
Neuroanatomical studies connecting
the circadian clock to photoperiodic
induction of diapause
Photoperiodic time measurement consists of four
components: (1) light receptors, (2) a photoperiodic
clock that distinguishes long nights/short days from
short nights/long days, (3) a photoperiodic counter
that accumulates information on successive long
nights/short days, and (4) output pathways that generate various photoperiodic phenotypes (Saunders
2002; Koštál 2011). During the induction of photoperiodic diapause in insects, the output pathway of
the photoperiodic clock signals the decision to arrest
development to neurosecretory cells in the brain and
other endocrine organs. Although much is known
about the light receptors and the hormonal signals
that underlie the photoperiodic diapause phenotype,
very little is known about either the clock that measures night length, or the counter mechanism. Given
that circadian clock cells and the signaling events
that initiate diapause are located within the brain,
neuroanatomical studies are a logical place to
search for physical connections between circadian
clock neurons and the photoperiodic clock underlying diapause induction.
Photoperiodic light receptors have been studied in
at least 19 insect species from six different orders,
and from these studies, it is apparent that reception
of light can occur either retinally and/or through
extraretinal receptors located in the brain or stemmata (Goto et al. 2010). The type of photoreceptors
that insects use to initiate photoperiodic diapause
does not correlate with phylogeny (Numata et al.
1997). For example, both Calliphora vicina and
Protophormia terraenovae are flies in the family
Calliphoridae, but the former uses the brain as its
site of photoreception (Saunders and Cymborowski
1996), whereas the latter uses its compound eyes
(Numata et al. 1997; Shiga and Numata 1997).
Possibly C. vicina also uses its compound eyes to
measure day length and initiate diapause, but this
was not tested by Saunders and Cymborowski.
Convergence and/or redundancy in the type of photoreceptors used to initiate diapause is a likely consequence of the multiple times that photoperiodic
diapause has evolved (Saunders 2012).
Although circadian clocks in insects are present in
multiple tissues, the brain-based clock is likely most
critical for circadian responses, but interestingly the
precise location of clock cells within the brain does
136
not seem to be well-conserved. In D. melanogaster,
staining for circadian clock neurons is localized to
three groups of lateral neurons and three groups of
dorsal neurons in the pars lateralis (PL) (HelfrichFörster 1995). The circadian clock in P. terraenovae
shows a similar arrangement to that of Drosophila
(Shiga and Numata 2009), but circadian clock neurons in cockroaches and crickets are localized in the
accessory medulla (Petri et al. 1995; Tomioka and
Matsumoto 2010). Circadian clock gene expression
in the silk moth, Antheraea pernyi, is confined to
eight neurosecretory cells in the pars intercerebralis
(PI) and PL, as probes for PER protein and per
mRNA colocalized in these cells (Sauman and
Reppert 1996). Similarly, the central circadian clock
in the monarch butterfly, D. plexippus, is localized to
four cells in the dorsolateral protocerebrum (PL),
while neurosecretory cells in the PI also express
PER and CRY1 (Sauman et al. 2005).
Hormones that control diapause are commonly
released from neurohemal organs known as the
corpus cardiacum (CC) and corpus allatum (CA).
Adult reproductive diapauses are characterized by a
failure of the CA to release juvenile hormone (JH),
which leads to arrested reproductive development.
Under short days, neurosecretory cells in the PL of
the brain of the Colorado potato beetle, Leptinotarsa
decemlineata, likely produce the hormone allatostatin
and block the CA from producing JH, which leads to
the diapause phenotype (Khan et al. 1986). In another species which has a photoperiodically-induced
adult reproductive diapause, P. terraenovea, nickel
back-filling demonstrated that neurosecretory cells
in the PI and PL innervate the CA and CC (Shiga
and Numata 2000, 2001). When the neurosecretory
cells in the PI were removed, females that would
normally develop their ovaries under long-day conditions displayed a diapause phenotype. In contrast,
when neurosecretory cells in the PL were removed,
most females continued to develop their ovaries even
when they were exposed to short-day diapause-inducing conditions. Although it is unclear from
these experiments whether removal of the PI and
the PL affects photoperiodic time measurement or
ovarian development directly, the results of this experiment suggest that in P. terraenovea, cells in the
PI produce a compound (perhaps allatotropin) that
is critical for ovarian development, whereas neurosecretory cells in the PL produce a compound (perhaps
allatostatin) that inhibits ovarian development.
More recently, Shiga and Numata (2009) have
investigated the site of photoperiodic time measurement in the brain of P. terraenovea. Circadian clock
cells that synthesize the protein pigment-dispersing
M. E. Meuti and D. L. Denlinger
factor (PDF) innervate neurosecretory cells in the
PL of P. terraenovea (Shiga and Numata 2009).
PDF is also co-expressed in many central circadian
clock cells in Drosophila and is an important output
of circadian clocks, thus suggesting an exciting
model that PDF accumulates under successive short
nights, reaches a critical threshold that signals neurosecretory cells in the PL to produce allatostatin,
and hence initiates diapause (Fig. 3). Indeed, ablation of PDF-expressing circadian neurons, which are
likely involved in photoperiodic time measurement,
resulted in a lower incidence of diapause in P.
terraenovea.
In A. pernyi and D. plexippus, two species of
Lepidoptera, the central circadian clock seems to be
localized within neurosecretory cells in the PL. In
addition, PER-expressing cells were found in the
CA of A. pernyi (Sauman and Reppert 1996) and
in the CC of D. plexippus (Sauman et al. 2005). A
CRY1-positive pathway connects D. plexippus circadian clock cells in the PL to both the light input
from the eyes and to neurosecretory cells in the PI.
These results demonstrate that clock neurons are
connected to regions of the brain that control diapause, and as clock genes are expressed in the neurohemal organs themselves suggest the possibility
that the cycling of clock genes in these organs may
also play an important role in initiating diapause.
Molecular studies on clock genes and
diapause
In D. melanogaster, flies that are null for the per
gene, and hence arrhythmic, are still capable of entering diapause (Saunders et al. 1989; Saunders
1990). Although the CPP is slightly shifted in pernull flies, the results suggest that per is not involved
in initiating the photoperiodic diapause in this species. These results, however, do not preclude the involvement of other clock genes, such as tim, in the
diapause response.
Two naturally varying alleles of tim are present in
D. melanogaster (Tauber et al. 2007); one that exclusively produces a short form of the mRNA (s-tim)
and one that produces both long and short forms
(ls-tim). The ls-tim form correlates with higher
levels of diapause in Europe, and hence is adaptive
in temperate climates. In a related study, Sandrelli
et al. (2007) demonstrate that the S-TIM protein
binds more tightly to CRY1 than to LS-TIM, such
that the shorter form is more readily degraded. As
LS-TIM has the potential to decrease light sensitivity
of the circadian clock, Sandrelli et al. (2007) postulated that the ls-tim allele would be adaptive in
Insect circadian clocks and photoperiodic diapause
Fig. 3 (A) Schematic representation of the brain of the blow fly,
Protophormia terraenovae (adapted from Shiga and Numata 2001,
2009), showing connections between PDF-positive, small lateral
ventral neurons (sLNv) that innervate neurosecretory cells in the
pars lateralis (PL). These neurosecretory cells innervate the corpora cardiacum (CC) and corpora allatum (CA) that produce
and/or release key hormones involved in development and diapause. (B) An hypothetical model showing the potential role of
PDF in initiating diapause. Under short days, high amounts of PDF
are transmitted to neurosecretory cells in the PL, promoting the
production of allatostatin, which prevents the CA from producing
juvenile hormone (JH), thereby resulting in adult reproductive
diapause. Under long-day conditions, PDF levels are low and the
neurosecretory cells in the PI produce allatotropin, which stimulates the CA to produce JH, leading to reproductive maturation.
northern latitudes where day length dramatically increases in the summer. However, the allelic distribution demonstrates that the s-tim allele is more
common in northern climates, and therefore allelic
variation of tim is not linked to the diapause response (Tauber et al. 2007). However, these results
might also be explained by the relatively recent
origin of the ls-tim allele, which arose 10,000
years ago in southeastern Italy (Sandrelli et al.
2007). This allele has been spreading throughout natural populations, and selection may be acting to
137
increase the allelic frequency of ls-tim in seasonal
European habitats (Kyriacou et al. 2008). Flies that
are tim-null are still capable of entering diapause, but
they lose their photoperiodic response, allowing a
portion of the population to enter diapause regardless of day length (Tauber et al. 2007). Therefore,
some components of the circadian clock, such as
tim but not per, may be involved in measuring day
length or modulating other features involved in the
induction of diapause in Drosophila.
As indicated above, Drosophila has a unique molecular clock that, unlike most other insects, lacks
cry2. Moreover, Drosophila has a weak diapause
that may be more akin to quiescence (Tatar et al.
2001). Thus, D. melanogaster is not the best organism to use for examining the relationship between
clock genes and the photoperiodic diapause response.
Also, as photoperiodic diapause has likely evolved
numerous times in insects, it is essential to examine
the connection between circadian clock genes and
diapause in diverse insect species to gain a more
comprehensive view of this interaction.
Research on the drosophilid fly Chymomyza costata implicates tim as a potential link between the
circadian oscillator and the photoperiodic induction
of diapause. Riihimaa and Kimura (1988) identified
a non-photoperiodic diapausing mutant strain of
C. costata, and Pavelka et al. (2003) mapped the
mutation to a deletion in the 50 leader sequence of
the tim gene. The deletion contains sites for transcription activation and regulatory motifs, including
the E-box promoter, which inhibit tim transcription,
resulting in low TIM protein levels in non-diapausing mutants (Stehlik et al. 2008).
The flesh fly, S. bullata, offers additional insight
into a potential role for per in the photoperiodic
diapause response. Goto et al. (2006) identified a
non-diapausing strain of flesh flies that also display
arrhythmic eclosion patterns. The lack of circadian
rhythmicity suggested a malfunction of the clock.
However, rather than showing suppression of per
or tim, both genes were more highly expressed in
mutant flies. A comparison of per sequences isolated
from wild-type, high and low diapausing strains of
S. bullata revealed that incidences of diapause were
higher in strains that had shorter PER C-terminal
regions, designated the C-terminal photoperiodic
(CP) region (Han and Denlinger 2009). Future research is needed to identify proteins that interact
with this region of PER as such proteins could be
involved in the photoperiodic induction of diapause.
Clock genes have also been implicated in the adult
photoperiodic diapause of the bean bug, Riptortus
pedestris. When R. pedestris are reared under
138
diapause-inducing short days or diapause-averting
long days per is expressed at nearly identical levels
(Ikeno et al. 2008). When per or cry2 expression was
knocked down using RNA interference (RNAi) both
the daily rhythm of cuticle deposition (a circadian
response) and ovarian arrest under short-day conditions (a photoperiodic response) were disrupted
(Ikeno et al. 2010, 2011a, 2011b). R. pedestris females
treated with per or cry2 double-stranded RNA
(dsRNA) produced a single dark layer of cuticle as
opposed to alternating dark and light layers in control insects. RNAi directed against per and cry2 also
caused females reared under short-day conditions,
which normally enter a reproductive diapause characterized by undeveloped ovaries, to develop their
ovaries. When RNAi was used to knock down cyc
or Clk, the opposite effect was observed: the bugs
produced a single bright layer of cuticle and failed
to develop ovaries even when reared under long-day
conditions (Ikeno et al. 2010; Goto 2013). This work
was repeated in male bean bugs with identical results: per and cry2 dsRNA caused short-day males
to avert reproductive diapause, while cyc dsRNA
caused long-day males to enter diapause (Ikeno
et al. 2011a). These results, and other targeted studies
on clock genes, demonstrate that circadian clock
genes are involved in photoperiodic induction of diapause, suggesting that insects may have co-opted
their ancestral circadian clocks during the evolution
of photoperiodic responses.
Pleiotropy, plasticicty, and programming
The problem of pleiotropy
Diapause initiation is a complex process, beginning
with perceiving light and interpreting photoperiod,
followed by storing this information and translating
it into hormonal signals that then lead to a complex
suite of physiological, morphological, and behavioral
changes that characterize the diapause phenotype.
Emerson et al. (2009) correctly pointed out that
the central circadian clock, or its individual genes,
may be interacting pleiotropically in any one or all
of these steps. This is noteworthy, as circadian clocks
regulate numerous behavioral and physiological processes, and there may very well be overlap between
circadian and diapause phenotypes. One recent and
elegant example of this (Bajgar et al. 2013) demonstrates in the linden bug, Pyrrhocoris apterus, that
CLK and CYC interact in the gut with JH to increase
expression of the clock gene Pdp1iso1 and suppress
expression of cry2 under long days. In contrast,
cry2 expression is elevated and Pdp1iso1 is suppressed
under short days. These clock genes are therefore
M. E. Meuti and D. L. Denlinger
acting to regulate the diapause status of the
P. apterus gut and are doing so independently of
their daily oscillations in circadian clocks. It is thus
challenging to determine where, and whether, circadian clocks (modular pleiotropy) or a clock gene
(gene pleiotropy) is exerting its effects on the initiation of photoperiodic diapause.
To empirically determine whether an individual
clock gene or the clock as a functioning module affects diapause, Emerson et al. (2009) encouraged researchers to examine the effects of multiple, single
clock gene knockouts. To date, the previously mentioned RNAi experiments in the bean bug are the
only experiments that have taken such an approach
(Ikeno et al. 2010, 2011a, 2011b; Goto 2013).
Knocking down per and cry2, which are both negative regulators of the circadian clock, stopped the
cuticle-deposition rhythm in the same phase (only
a dark layer was produced), and both caused the
same effect: aversion of diapause under short-day
conditions (Ikeno et al. 2010, 2011a, 2011b). In contrast, knocking down cyc or Clk, which are positive
regulators of the circadian clock, arrested the cuticledeposition rhythm in a different phase (only bright
layers were produced), and both male and female
bugs entered diapause under long-day conditions
(Ikeno et al. 2010, 2011a, 2011b; Goto 2013). This
is strong evidence that the circadian clock, as a functioning unit, is involved in the diapause response in
R. pedestris because circadian genes that have the
same role in the clock exert the same effect on
adult reproductive diapause.
Yet, the question remains: where in the process are
these clock genes affecting the diapause response?
The experiments by Ikeno et al. (2010) offer further
insight: the long-day diapause induced by RNAi
against cyc could be terminated by applying a JH
analog to the cuticle. This demonstrates that knocking down cyc affects diapause upstream of JH release
from the CA, but it is still unclear whether the
central R. pedestris circadian clock is involved in
photoreception, measurement of day length, or neurosecretory signaling to the CA.
Plasticity in the diapause program
As previously mentioned, diapause appears to have
evolved numerous times in insects. This is supported
by the number of life stages that enter diapause
within given taxa, as well as by the variation in the
types of cues, photoperiodic and otherwise, that insects use to enter diapause. The origin of diapause in
insects is still unclear, as more primitive animals,
such as copepods (Hairston and Munns 1984), also
Insect circadian clocks and photoperiodic diapause
enter diapause, and the capacity for diapause is extremely widespread. The diapause of some temperate
insects can be traced to the tropics where diapause is
also common (Tauber and Tauber 1981; Denlinger
1986). As several tropical insects, such as flesh flies,
colonized temperate environments natural selection
altered various aspects of the underlying diapause
phenotype, most notably by adding the ability to
use photoperiod to predict seasonal changes in the
environment. In the tropics, where changes in day
length are slight, insects use cues other than photoperiod, such as temperature, rainfall, food quality,
and population density, to initiate non-photoperiodic diapause (Denlinger 1986). As insects colonized
higher latitudes, mechanisms for measuring day
length were acquired. For example, tropical populations of the pink bollworm, Pectinophora gossypiella,
have a lower incidence of diapause but are more
likely to enter diapause in response to non-photoperiodic changes in food and temperature relative to
populations from higher latitudes (Ankersmit and
Adkisson 1967). In contrast, as other species moved
into subtropical or tropical regions from temperate
zones, they lost their ability to enter a photoperiodically-induced diapause (e.g., embryonic diapause in
Ae. albopictus; Lounibos et al. 2003).
Additionally, flexibility in features of the diapause
program, such as critical day length and duration
and depth of diapause, has allowed insects to colonize new areas and has contributed to their evolutionary success. The strong positive correlation
between CPP and latitude/elevation in W. smithii
nicely demonstrates the importance of variation in
this trait (Bradshaw et al. 2003b, 2006). Although
the CPP differs among populations of W. smithii,
the period of clock gene cycling remains constant
although there are some changes in amplitude of
cycling in the clock genes (Mathias et al. 2005).
The authors argued that it would be maladaptive
to couple circadian clocks, which need to maintain
a stable period, to a photoperiodic diapause response
that needs to remain flexible (Bradshaw et al. 2003b;
Bradshaw and Holzapfel 2007).
Reconciling the need for a flexible diapause program
with a stable clock
As suggested above, circadian clocks likely evolved
early and, due to their adaptive significance
(Sharma et al. 2003), the DNA sequences of clock
genes have been well-conserved. There are, however,
significant changes in the composition of insect
clocks that occurred during evolution, such as the
loss of cry2 in Drosophila, the loss of cry1 in the
139
flour beetle, T. castaneum, and the loss of both tim
and cry1 in the honey bee, Apis mellifera and other
hymenopterans (Yuan et al. 2007; Sandrelli et al.
2008). Yet, changes in the gene composition of circadian clocks did not change the inherent circadian
periodicity (i.e., their circadian oscillators continue
to cycle with endogenous periods that range from
22 to 25 h in constant darkness), thus demonstrating the diverse structural possibilities of the clock.
The fact that the endogenous period of most circadian oscillators () is not equal to 24 h indicates that
insects have to continually reset their circadian pacemakers to match the periods of their environments.
Therefore, circadian pacemakers can reset, or entrain
to diverse inputs, such as temperature (Zimmerman
et al. 1968) and food (Frisch and Aschoff 1987), as
well as to light:dark cycles. Other features of circadian clocks in insects, such as their ability to measure
night length by resetting to ‘‘lights-off cues,’’ may
have predisposed the clock to measure photoperiod
(Saunders 2009). Pittendrigh and Daan (1976) used
sophisticated models to demonstrate that when
6¼ 24 h, animals are able to reset their endogenous
circadian pacemakers to ‘‘lights-on’’ (when 424 h),
or ‘‘lights-off’’ (when 524 h) cues over a broader
range of photophases than when ¼ 24 h. Therefore,
having an endogenous circadian period that differs
from 24 h allows temperate insects to maintain circadian oscillations in environments in which day
length changes dramatically throughout the year.
Circadian clocks control diverse behaviors, and we
are now gaining a better appreciation for the extent
of physiological and molecular processes that are also
under circadian control. A recent transcriptome analysis of the malaria mosquito, A. gambiae, revealed
that at least 15.8% of the genes are under either
circadian or diel control (Rund et al. 2011).
Genes involved in transcription and translation,
metabolism, detoxification, olfaction, vision, and
immunity all show daily expression profiles both
under light:dark cycles and continuous darkness.
Similarly, studies of Drosophila demonstrated that
hundreds of transcripts are under the control of circadian clock genes (Claridge-Chang et al. 2001;
McDonald and Robash 2001).
In contrast to circadian clocks, the photoperiodic
diapause response has evolved numerous times in
insects, and many diapause characteristics remain
plastic and variable both within and between populations. Yet, the circadian clocks that operate among
populations of insects with diverse photoperiodic
diapause responses must and do allow insects to
maintain circadian oscillations over the 24-h environmental period. Therefore, it is necessary to
140
think of ways in which circadian clocks can be coupled to photoperiodic time measurement such that
the stability of circadian clocks and flexibility of the
diapause response are maintained. Goto (2013) and
Tagaya et al. (2010) summarize several arguments for
how this might be accomplished. Under the external
coincidence model, insects from different populations can alter timing of their light sensitive stage
(’i), such that populations from higher latitudes
would be expected to have a later ’i than that of
southern populations, thereby accounting for differences in CPP. Different populations may also have
different thresholds for a diapause-inducing substance, or different rates of synthesis or degradation
of such a compound (Tagaya et al. 2010; Goto 2013).
These scenarios for altering CPP for the induction of
diapause do not require changes in the central circadian clockwork, and we argue that such possibilities
could explain how insects have co-opted their existing circadian clocks to measure photoperiod and
properly time the initiation of diapause.
Conclusions and future directions
Although more than 75 years have elapsed since
Erwin Bünning proposed that photoperiodic responses are linked to endogenous circadian clocks,
we still lack a mechanistic understanding of the central circadian clock’s involvement in photoperiodic
diapause. Yet, a wide variety of classical experiments,
such as Nanda–Hamner resonance and Bünsow night
interruption protocols, point to a circadian basis for
photoperiodic induction of diapause in nearly all
insect species that have been studied. Additionally,
several insect species with mutations in their circadian clock genes show abnormal diapause responses,
including either changes in their CPP as in per-null
Drosophila, or failure to enter diapause as in the CPper mutant of S. bullata, and deletion of E-box sequences in the tim promoter in C. costata. Targeted
RNAi experiments have demonstrated that the central circadian clock, as a functioning unit, is necessary for photoperiodic diapause initiation in the bean
bug, R. pedestris. Similar approaches in additional
species are needed to examine whether individually
knocking down multiple positive and negative circadian regulators elicits the same effect on the induction of diapause. Experiments of this type will enable
us to determine whether the central circadian clock
is functioning as a module or whether individual
clock genes are acting independently in initiating
the diapause program.
Although it remains unclear how insects connect
their circadian clocks to photoperiodic time
M. E. Meuti and D. L. Denlinger
measurement without compromising the integrity
or stability of their circadian oscillators, several possibilities have been suggested (Tagaya et al. 2010;
Goto 2013). Future research is needed to determine
whether and how insects shift their light sensitive
stage ’, and also to determine whether a clock-derived compound is involved in photoperiodic diapause initiation. Currently, we do not have solid
evidence for such a substance, although PDF is an
enticing candidate, especially in light of Shiga and
Numata’s (2009) work with pdf-expressing neurons
in P. terraenovea. Such neuroanatomical studies in
additional insects can be expected to yield further
insights into physical connections between the circadian clock and the photoperiodic diapause response.
Bradshaw and Holzapfel (2007) urged researchers
to abandon studies that examined the effects of individual clock genes on the diapause response in
favor of forward genetic techniques. Although we
disagree that targeted research on clock genes has
little promise, we do believe that researchers should
also employ forward genetic techniques to identify
non-circadian genes involved in photoperiodic diapause responses. As photoperiodic diapause has
evolved multiple times in insects, and as the core
circadian clock also differs among insect taxa, it is
quite likely that circadian clocks play different roles
in the photoperiodic responses of different species.
We urge the use of every experimental technique in
our arsenal, from continued classical experiments
through RNAi and RNAseq, to determine the exact
role of circadian clocks in photoperiodic induction
of diapause in a wide range of insect species.
Acknowledgments
We thank the reviewers and Harold Heatwole, editor
of this journal, for their thoughtful consideration
and helpful critique of this article.
Funding
This work was supported in part by the National
Institutes of Health Grant 2R56-AI058279 and the
NSF Graduate Research Fellowship Program.
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