university of groningen latitudinal differences in the

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Latitudinal differences in the circadian system of Nasonia vitripennisFloessner, Theresa

DOI:10.33612/diss.102037680

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39

Chapter 3 Increased circadian light sensitivity in a northern

line of Nasonia vitripennis

Abstract

Light sensitivity of the circadian system has been proposed to decrease with latitude. We tested

this hypothesis in Nasonia vitripennis, a parasitoid wasp with a world-wide distribution range.

Using a northern and a southern European line, we generated a series of full dose-response

curves with varying light pulse duration and intensity. From this we established at which light

intensity circadian light resetting changes from weak (type-1) to strong (type-0) and we deduced

total photon-dose response curves. Both approaches show that the circadian system of N.

vitripennis from the north is more light sensitive than from the south. This higher light

sensitivity increases the range of entrainment and compensates for the larger deviation of

circadian free-running period from 24h, which is considered to serve latitudinal adaptation of

the photoperiodic system.

Theresa S.E. Floessner1, Mirjam Bakker1, Tom Woelders1†, Domien G.M. Beersma1, Roelof A.

Hut1

1Chronobiology Unit, Neurobiology expertise group, Groningen Institute for Evolutionary Life

Sciences, University of Groningen, the Netherlands.

†Current Address: Division of Neuroscience & Experimental Psychology, School of Biological

Sciences, Faculty of Biology, Medicine and Health, University of Manchester, United Kingdom

Chapter 3

40

Introduction

Most organisms are exposed to seasonal changes in the environment, characterised by

differences in temperature and photoperiod (duration of the daily light phase). Seasonal

amplitude differs in a latitudinal gradient due to the Earth’s tilt of 23.5° and its elliptical path

around the sun. Extreme latitudes, close to the poles, experience stronger seasonal variation in

both temperature and photoperiod than latitudes closer to the equator (Hut et al. 2013). At any

given date, the lower solar position at higher latitudes results in lower temperatures due to

reduced solar radiation. However, spring and summer photoperiods are longer at higher

latitudes, resulting in longer influx of solar radiation. Together this leads to complex

asymmetrical change of the annual ellipsoid photoperiod-temperature relationship with

increasing latitude (Hut et al. 2013). Organisms have adapted the timing of their annual winter

survival strategies, including migration, hibernation, torpor and diapause, to these latitudinal

changes in the photoperiod-temperature relationship. Most organisms, including plants, insects,

birds and mammals, use photoperiod (Lees 1955) as a reliable proxy for upcoming seasons as

it is a stable cue over millions of years, unlike temperature for instance (Withrow 1959; Frisch

1960; Aschoff 1965; Menaker 1971; Bradshaw et al. 2013) thereby enabling deeply rooted

evolutionary adaptation. Insects can survive harsh winter conditions in a quiescent state called

diapause, which is genetically determined and characterized by reproductive arrest, suppressed

metabolism and high tolerance to environmental stress (Tauber et al. 1986). Depending on the

species, it can occur in all stages of the life cycle (Tauber et al. 1986). Latitudinal changes in

the annual ellipse-like relationship between temperature and photoperiod predicts variation

over the north-south axis in diapause inducing photoperiod (critical photoperiod). The predicted

latitudinal increase in critical photoperiod is consistent with data from many insect species

(reviewed in Hut et al. 2013).

The circadian clock regulates physiology and behaviour on a daily basis and uses mainly

the light-dark cycle to entrain to the 24-h environmental day. The circadian clock is driven by

interlocked transcriptional translational feedback-loops (Hall 2003, Hardin 2005), consisting of

several clock gene - clock protein interactions (Gallego and Virshup 2007) that either activate

or inhibit their own transcription or the transcription of other clock genes. It is likely that the

molecular circadian clock work is involved in seasonal time measurement. Adaptation to

latitude may thus involve evolutionary changes in the genetic make-up of the circadian system,

including its light sensitivity. In Drosophila melanogaster populations, length polymorphisms

of the clock gene period (per) revealed a latitudinal cline in a threonine-glycine (Thr-Gly)

repeat encoding region, where longer variants occur at higher latitudes (Costa et al. 1992)

leading to the suggestion that its function in thermostability contributed to a circadian

phenotypic selection (Sawyer et al. 1997). Furthermore, a study by Tauber et. al., 2007 also in

Drosophila melanogaster showed polymorphisms in another important clock gene timeless

(tim), encoding a light-sensitive circadian regulator (Suri et al. 1998, Yang et al. 1998). Also

here a latitudinal cline was identified along a European north-south axis in allelic frequency of

an alternative start initiation variant (Rosato et al. 1997). Additionally, diapause induction

followed a similar latitudinal cline, with induction earlier in the year in the northern population

than in the southern, reinforcing the notion of involvement of the circadian system in seasonal

time measurement (Tauber and Zordan 2007).

Circadian light sensitivity in Nasonia

41

Latitudinal variation in photoperiodism was documented already in detail by Danilevskii

(1965). He gave an overview of a positive correlation between latitude and critical photoperiod.

In a more recent overview, most species studied so far show indeed positive correlation between

latitude (as distance from the equator) and critical photoperiod (Hut et al. 2013), including

Nasonia vitripennis (Paolucci et al. 2013). The females of this parasitoid wasp induce larval

diapause in short photoperiods (Saunders 1965a, 1966a). QTL analyses of N. vitripennis

populations originating from different European latitudes identified two candidate genomic

regions involved in diapause induction (Paolucci et al. 2016) These regions include the clock

genes period (per) and cycle (cyc) (both chromosome 1) and cryptochrome (cry)

(chromosome 5). Furthermore, an analysis on polymorphisms of per showed a clinal

distribution of two main haplotypes of per correlating with the latitudinal cline in diapause

induction (Paolucci et al. 2013, 2016). Per RNAi in Nasonia confirmed the role of the circadian

clock in photoperiodism, as knock down of per causes a reduction of diapause incidence under

short photoperiod (Mukai and Goto 2016). The strong link between such circadian properties

and photoperiodism suggests an essential involvement of the circadian system in

photoperiodism, seasonal rhythms and latitudinal adaptation.

Pittendrigh and Takamura (1987, 1989) described a latitudinal cline in critical

photoperiod in Japanese Drosophila auraria and measured phase response curves of fly

populations from different locations covering a 8° latitudinal range. Using identical light

stimulations, D. auraria from the south responded with larger circadian phase shifts than flies

from the north. This decrease in circadian light sensitivity with increasing latitude was

hypothesized to be a functional circadian pacemaker adaptation. Northern populations would

have developed stronger pacemakers, with larger circadian amplitude and smaller light

responses, to avoid circadian amplitude reduction under long photoperiods or continuous light

conditions (Pittendrigh et al. 1991). However, other circadian indices measured over longer

latitudinal rages in other Drosophila species, consistently show a decrease in circadian

amplitude (Hut et al. 2013)

Here we aim to test the reduction of circadian light responses in the parasitoid wasp

Nasonia vitripennis, which has a world-wide distribution (Werren and Loehlin 2009). Unlike

Drosophilids, N. vitripennis is a diurnal insect, which maintains circadian rhythmicity under

continuous light conditions. In addition, its latitudinal cline of critical photoperiod and the

involvement of the circadian system in photoperiodic diapause induction is well established

(Saunders 1974; Paolucci et al. 2013).We measured circadian phase response curves

(determined by locomotor activity recordings) in a northern line (collected in Oulu, Finland,

65.01°N) and a southern line (from Corsica, France, 42.04°N), using light stimuli of various

durations and intensities. By comparing these combinations we generate insight in the circadian

response landscape and the underlying process of light integration of the circadian system. We

are identifying reciprocity of intensity and duration by constructing a general photon dose

response curve for circadian light responses within each strain and sex. Population and sex

specific photon dose response curves allow for comparing precise estimations of circadian light

sensitivity between the northern and southern lines.

Chapter 3

42

Materials & Methods

Experimental lines and maintenance

The experiments were performed with the parasitoid wasp Nasonia vitripennis; with lines

originating from Oulu, Finland (65.01°N) and Corsica, France (42.04°N) (Paolucci et al. 2013).

Most experiments were conducted with one isofemale line from Oulu (northern line) and one

isofemale line from Corsica (southern line) established by Paolucci et al. 2013. Other lines from

the Oulu and Corsica region were used for control experiments. All lines were reared in a

temperature and humidity controlled climate chamber (20 ±1°C, 50-55% RH) in a light-dark

cycle of 16-h of light (646 lux) and 8-h of darkness per day (LD 16:8) to prevent diapause

induction. All individuals were offspring from separately housed females that were presented

to Calliphora spp. pupae as hosts.

Locomotor activity measurements

Circadian phase shifts to single light pulses at different times of day were conducted in an

Aschoff type II experiment (Aschoff 1965). All animals, independent from the time of light

pulse, receive the same light-dark cycle (LD) of 16h:8h during the first five consecutive days,

followed by constant darkness (DD) where the light pulse was given after two days of DD;

different groups of individuals received group specific single light pulses at 12 different time

points in 2-h intervals. As circadian output we recorded locomotor activity of individuals by

using the Drosophila Activity Monitoring System (DAMS, by TriKinetics, Waltham, USA). To

detect the new phase after the light stimulation the recording continued for at least seven

consecutive days in constant darkness and was compared to individuals which did not receive

any light pulses. For the activity recording, individuals (eight per experimental group and

condition) of the age of three to five days, fertilized females and males, were transferred into

activity tubes (6.5 x 0.5 cm), filled one quarter with agar food (30% sucrose, 1.5% agar, 0.15%

nipagin) and closed with a cotton plug on the other end. The tubes fit into recording monitors

(32 tubes in one monitor) that recorded activity per minute of each individual separately by

infrared light beams that were interrupted when a wasp crossed it. The monitors were placed

into light-tight boxes (23 x 14 x 32 cm), in 18°C (±1 °C) and 50-55% RH. Each light-tight box

was illuminated with one LED light source (Neutral White 4000K, PowerStar, Berkshire) of

providing maximally 2.10 · 1015 photons·cm-2·s-1 (high light intensity). To decrease light

intensity we inserted neutral density filters into the light-tight boxes two hours before “light

off” of the last LD cycle. Filters reduced light intensity to 2.62 · 1014 photons·cm-2·s-1

(intermediate light intensity) and 9.37 · 1013 photons·cm-2·s-1 (low light intensity).

Determination of Phase Shifts

Phase shifts were determined in ChronoShop (Spoelstra et al. 2004) individually by comparing

old phase before the light pulse, and new phase, after the light pulse (excluding the first two

transient days after the light pulse). As a phase marker for the activity rhythm we used centre

of gravity (Kenagy 1980). In average 12% of individuals were excluded when the visual


43

inspection of the actograms showed arrhythmicity (usually Lomb-Scargle dPn value<15) or

abnormal activity patterns. The timing of the light pulse was calculated relative to the light dark

cycle and the phase shift was calculated to a dark control for each sex and strain specifically.

Because phase shifts may exceed 12 h, we used circular averages by calculating the average

vector on a circular 24-h scale. Phase response curves were plotted as average phase shift (h)

against time of mid pulse (ZT, h). Classification into strong (type 0) or weak (type 1) phase

resetting was obtained by visual inspection by triple plotting the abscissa and ordinate (both

being circular time scales) of the phase transition plots (new phase vs. old phase). This yields

clear horizontal patterning (slope=0) of the data in the case of type 0 phase transition curves

(PTCs) and slant patterning (slope=1) in the case of type 1 PTCs. We obtained clear horizontal

(type 0) or slanted (type 1) patterns, allowing for clear determination of strong or weak resetting

for all obtained phase shift curves.

Light pulses of various duration (0.3, 1, 4, 8, 16 h) and intensity (9.37*1013 (low); 2.62 ·

1014 (intermediate); 2.10 · 1015 (high) photons·cm-2·s-1) were applied. By multiplying duration

and intensity, we calculated photon dose (in photons·cm-2) for each combination of intensity

and duration. To calculate photon dose response curves within each line and sex, each PRC was

collapsed into a single value by integration towards the 0h phase shift axis between ZT0 and

ZT16. This integral coincides with the light phase during previous entrainment and allowed for

selective evaluation of entraining circadian light responses. Although there is no clear

distinction between large advances and large delays in type 0 phase response curves, our

method of restricted integration allowed us to avoid the discontinuous transition between

advances and delays which would otherwise prevent unambiguous calculation of the integral.

Photon dose response curves through these integral values were fitted using a modified Naka-

Rushton equation (Hut et al. 2008).

Results

PRCs with different light pulse durations

To determine circadian light sensitivity in northern and southern Nasonia vitripennis lines

circadian phase shift responses to light pulses of various duration were quantified. Average

circadian phase shifts (h) were plotted against mid-pulse phase (ZT, h), generating so-called

phase response curves (PRCs). Classification of PRCs in strong (type 0) or weak (type 1)

resetting was unambiguous using the triple-triple PTC plotting technique as described in the

methods. This may also have resulted in phase shifts exceeding 12h, which was allowed for

continuity reasons in the case of type 0 PRCs.

Females responded to 0.3 h and 1 h light pulses with small phase shifts, whereas in 4-h

light pulse duration animal from the northern line mainly shift their phase with advances of

several hours during the subjective day. Light pulses during the subjective night caused phase

delays (Fig. 1). Similar responses are observed for longer light pulse durations (8-h and 16-h).

Also in the southern line in the longer light pulse condition the type-0 PRCs are characterized

by big phase advances in the subjective day but the crossover point occurred earlier in the day

Chapter 3

44

at ZT12 in the second half of the subjective day which caused a broader delay phase (Fig.1,

Table 1).

Northern males in 0.3 h light pulses showed mainly phase advances for all measured time

point besides ZT20 (Fig. 2). Light pulses of 1-h caused strong resetting with biggest phase shifts

at ZT0, phase advances in the subjective day and phase delays in the subjective night. This

holds for 1-h and 4-h light pulses, in 8-h and 16-h light pulses the crossover point advanced

towards ZT12, consequently we see a broader delay phase. Males from the southern line showed

in 0.3-h and 1-h light pulse durations nearly no phase shifts (Fig. 2). In 4-h light pulses we see

phase advances in the first half of the subjective day, phase delays in the second half of the

subjective night, and a dead zone in the second half of the subjective day. In 8-h and 16-h light

pulse condition the curves pattern are very similar to the ones from the northern males; biggest

shifts at ZT0, phase advances in the first half of the subjective day followed by a broader delay

phase (Fig. 2).

Overall we see that longer light pulses generate larger phase shifts, with maximum

response at ZT0 (Fig. 1, 2 & Table 1). The transition from weak (type 1 PRC) to strong (type 0

PRC) responses occurs in the northern line at shorter light pulse durations than in the southern

line, indicating higher circadian light sensitivity in the northern line. Northern females show

strong resetting at 4-h light pulses whereas the southern females still show a weak resetting

response. The females’ transition from weak to strong circadian resetting occurs at 4 and 8 hour

light pulse duration in northern and southern wasps, respectively (Fig. 1 & Table 1). In males,

the transition from weak to strong resetting occurs at 1 and 4-h light pulse duration in northern

and southern wasps, respectively (Fig. 2 & Table 1).


45

Figure 1 Phase response curves (PRCs) from individually recorded Nasonia vitripennis females from

northern and southern Europe. Light pulses of different durations (indicated on the left side of each graph-pair)

and high light intensity were applied. Circadian phase shifts (h) are plotted against time of mid-light pulse (ZT, h).

Open circles represent individual phase shifts, connected solid circles represent circular averages. Shorter light

pulse durations caused smaller phase shifts (type 1 PRC) whereas longer light pulses have led to bigger phase

shifts (type 0 PRC). A transition from weak (type 1) to strong (type 0) PRCs occurred in the northern line with

light pulses of 4 h or longer and in the southern line with light pulses of 8 h or longer.

Chapter 3

46

Figure 2 Phase response curves (PRCs) from individually recorded Nasonia vitripennis males from

northern and southern Europe. Light pulses of different durations (indicated on the left side of each graph-pair)

and high light intensity were applied. Circadian phase shifts (h) are plotted against time of mid-light pulse (ZT, h).

Open circles represent individual phase shifts, connected solid circles represent circular averages. Shorter light

pulse durations caused smaller phase shifts (type 1 PRC) whereas longer light pulses have led to bigger phase

shifts (type 0 PRC). A transition from type 1 to type 0 PRCs occurred in northern line with light pulses longer than

1 h and in the southern line with light pulses longer than 4 h.


47

Table 2 Overview of weak and strong resetting to different light pulse durations (0.3-h, 1-h, 4-h, 8-h, 16-h) of

high light intensity in females and males from the northern and southern line. Responses became stronger (type 0)

with longer light pulse durations. The transition from weak (type 1) to strong response occurs at shorter stimulus

durations in the northern line than in the southern line. In both lines, males transitioned to strong resetting at shorter

pulse durations than females.

PRCs with different light intensities

To study differences of circadian light response to different light intensities we performed two

additional sets of phase response curves with low and intermediate light intensities for 1-h pulse

duration and for 4-h pulse duration. Female and male Nasonia wasps from the same northern

and a southern line are compared.

PRCs were constructed using 1-h light pulses at three light intensities (low, intermediate

and high). High intensity pulse responses were replotted from the first experiment (Fig. 1 &

Fig. 2). The results show a reduction in response with decreasing light intensities (Fig. 3 & Fig.

4). Although females from both lines showed type 1 PRCs with weak resetting, individuals

from the north expressed bigger phase shifts than the ones from the south in all three light pulse

intensities (Fig. 3 & Table 2). Northern animals showed small phase delays in the early

subjective day and during the subjective night in all three light conditions; in low and

intermediate intensity conditions we see phase advances in the second half of the subjective

day, that vanish in high light intensity condition into a dead zone of nearly no shifts (Fig. 3). In

males the differences between the lines were even clearer (Fig. 4). At all three light pulse

intensities the northern individuals showed type 0 PRCs with big phase advances in the early

subjective day and phase delays in the subjective night whereas the southern line show type 1

PRCs with nearly no phase shifts at all (Table 2).

north south north south

0.3 1 1 1 1

1 1 1 0 1

4 0 1 0 0

8 0 0 0 0

16 0 0 0 0

pulse duration (h)female male

Chapter 3

48

Figure 3 PRCs using 1-h light pulses at three different intensities from individually recorded Nasonia

vitripennis females from northern and southern Europe. Low, intermediate and high light intensity pulses

(indicated on the left side of each graph-pair). Circadian phase shifts (h) are plotted against time of mid-light pulse

(ZT, h). Open circles represent individual phase shifts, solid circles and lines represent circular averages. Both

lines show weak resetting (type 1 PRCs), while in general the northern line expresses bigger phase shifts than the

southern line in all three light conditions.


49


vitripennis males from northern and southern Europe. Low, intermediate and high light intensity pulses


(ZT, h). Open circles represent individual phase shifts, solid circles and lines represent circular averages. In all

three light intensities the northern line shows strong resetting (type 0 PRCs) and the southern line weak resetting

(type 1 PRCs). Within the lines there are no big differences between the different light conditions.

Using 4-h light pulses we observe that northern line females responded to low intensity

light pulses only with phase shifts in the subjective day and with biggest advances at ZT0 that

damped out towards the end of the subjective day (Fig. 5). A dead zone can be identified

between ZT12 and ZT18 with no phase shifts. Only small phase delays were observed during

the subjective night, which increased with increasing light intensity (Fig. 5). At high light

intensities the dead zone disappeared, phase advances occurred throughout the entire subjective

day until ZT18. Phase delays are observed in the second half of the subjective night. Females

from the southern line showed small phase advances in the early subjective day and small phase

delays during the subjective night. This holds for all three light intensities (Fig. 5).

Males from both lines showed strong resetting in all three light pulse intensities using 4-h

light pulses (Fig. 6). The northern ones showed big phase advances in the first half of the

Chapter 3

50

subjective day, in the second half a dead zone with small phase delays during the subjective

night. Similar responses are observed in intermediate light intensity conditions. The high

intensity light pulses (replotted from Fig. 2) led to big phase advances in the early subjective

day with a descending plateau phase during the late subjective day and early subjective night.

Stronger phase delays are found at the end of the subjective night. Southern line males

responded to low intensity light pulse with smaller phase advances than the northern males but

with bigger phase delays in the second half of the subjective day and during the subjective night

(Fig. 6).


vitripennis females from northern and southern Europe. Low, intermediate and high light intensity pulses


(ZT, h). Open circles represent individual phase shifts, solid circles and lines represent circular averages. The

northern line responds with strong resetting, type 0 PRCs, to all three intensities, whereas the southern shows type

1 PRCs to all three intensities.


51


vitripennis males from northern and southern Europe. Low, intermediate and high light intensity pulses


(ZT, h). Open circles represent individual phase shifts, solid circles and lines represent circular averages. Both

lines showed strong resetting (type 0 PRC); the northern line showed even bigger phase shifts, especially the phase

advances, than the southern line.

In summary, circadian behavioural phase shifting responses decreased with reducing light

intensity (Fig. 3 & Fig. 6). Northern lines (female and male) expressed bigger phase shifts than

the southern lines (compare Fig. 3 & Fig. 5 for females and Fig. 4 & Fig. 6 for males). In 1-h

light pulse duration females from both populations show weak resetting as well as males from

the south whereas northern males show strong resetting for all three light intensities (Fig. 3 &

Fig. 5 & Table 2). In the 4-h light pulse duration, northern females showed type 0 PRCs, for all

three light intensities, whereas the females from the southern line showed type 1. Males from

both lines responded to all light intensities with type 0 PRCs (Table 2).

Chapter 3

52

Table 3 Overview of weak (type 1) and strong (type 0) circadian resetting to 1-h and 4-h pulses of low,

intermediate and high light intensity, for males and females from the northern and southern line. Main differences

are found between light pulse durations where northern females and southern males increased their responses from

weak to strong when light pulse duration increased. Light intensity range was not large enough to elicit differences

in response type within each group.

Biological replicate-experiments were performed using 1-h and 4-h light pulse durations

at four different times (ZT0.5, ZT2, ZT16.5, and ZT18), in females and males from northern

and southern lines and three different light intensities. In these 48 conditions we did not find

significant differences (6/48 =12.5%; χ 2=5.4, p>0.9). Further we tested other isofemale lines;

two from the northern and two from the southern origin, using a 1-h light pulse duration with

the high light intensity at five different times (ZT0.5, ZT4.5, ZT8.5, ZT16.5, and ZT20.5), in

females and males. In a minority of conditions, the northern line showed quantitatively different

phase shift responses from the data presented here (7/20=35%; χ 2=36, p<0.02). The southern

line showed different results in only three out of 15 conditions (20%; χ 2=6.75, p>0.9).

Photon dose response curves

To evaluate pulse duration and light intensity results together and in relation to each other, we

calculated photon dose (in photons·cm-2) for each type of light stimulation by multiplying light

intensity (in photons·cm-2·s-1) by pulse duration (in s).

The single integrated value obtained for each PRC allows for constructing circadian

photon dose response curves for each sex and strain separately (Fig. 7). Comparing these dose

response curves we see that females of both lines show larger maximal phase shift amplitudes

than males indicating higher total light responses during the subjective day than males. In

addition, dose response curves in northern lines are shifted to lower light intensities compared

to southern lines, indicating higher light sensitivity in both northern females and males (Fig. 7,

dashed lines).

light intensity north south north south north south north south

low 1 1 0 1 0 1 0 0

intermediate 1 1 0 1 0 1 0 0

high 1 1 0 1 0 1 0 0

1-h light pulse 4-h light pulse

female male female male


53

Figure 3 Photon dose response curves for females and males from northern and southern lines. Each point

is the integration of a phase response curve (area-under-curve over ZT0-16). Integrated values originate from light

pulse duration experiments at high light intensity (Fig. 1 & Fig. 2, grey), light pulse intensity experiments using

1 h or 4 h light pulses (Fig. 3 & Fig. 6, black), or both (grey filled black circle). Dashed lines indicate curve

inflection points, corresponding to the inverse of light sensitivity.

Chapter 3

54

Table 3 Photon dose response curve parameters for each strain and sex. The fitted sigmoidal function is a

modified Naka-Rushton equation of the type: y = min + (max - min) / (1 + 10^(slope*(x - xinflection))), where x is

the light intensity in log10 photons·cm-2·s-1. Sensitivity is calculated as 1/xinflection. Model parameters were estimated

using an R-based non-linear regression procedure allowing simultaneous parameter estimation for each strain and

sex. The final model included both sex and strain as variables for maximum, slope and inflection point estimation

(F10,26=4.65, p<0.001).

Conclusion and Discussion

To study latitudinal variation in circadian light response, we measured phase response curves

in Nasonia vitripennis females and males from a northern (65.01°N) and southern (42.04°N)

population. Light stimulations at different circadian times, varying in duration and intensity,

revealed increased circadian light sensitivity in northern strains (Table 1 & Table 2 & Fig. 7).

In addition, the circadian system of males has a higher sensitivity to light than females, although

females were found to have a higher maximum response than males (Table 1 & Table 2 &

Fig. 7).

Our conclusion does not confirm findings in Drosophila aurora (Pittendrigh and

Takamura 1987) but seems indirectly supported by decreasing overt circadian amplitude with

latitude in D. melanogaster (Allemand 1976), D. subobscura (Lankinen 1993), and D. littoralis

(Lankinen 1986a). Pittendrigh and Takamura (1987) argued that latitudinal increase in

photoperiod (and eventually continuous light) would decrease the amplitude of the circadian

system. Indeed it has been ubiquitously observed in many species that long photoperiods can

reduce circadian amplitude up to arrhythmicity. Based on their measurements in D. aurora over

a small latitudinal cline, Pittendrigh (Pittendrigh and Takamura 1987; Pittendrigh et al. 1991)

argued that evolutionary adaptation would select for larger circadian amplitude with increasing

latitude to compensate for the amplitude reducing effect of longer light exposure. Consequently,

increasing amplitude of the circadian pacemaker would result in resilience to the phase shifting

effect of the stimulus, measured as reduced light sensitivity with increasing latitude. However,

other authors (Allemand 1976, Lankinen 1986, 1993) showed in other Drosophillids, over a

much larger latitudinal range, that circadian amplitude in overt rhythms decreases with latitude.

north south north south

min 0 0 0 0

slope

parameter0.44 0.44 0.44 0.44

max 234 191 115 71

inflection

point18.8 19.9 16.3 17.4

Sensitivity 0.053 0.050 0.061 0.057

model

parameters

female male


55

If these amplitude indices indeed allow us to make valid statements about the amplitude

of the underlying circadian pacemaker, than it should be concluded that amplitude of the

circadian pacemaker decreases with increasing latitude. Oscillator theory would than predict

that light sensitivity would increase with increasing latitude, since less robust, lower amplitude

circadian pacemakers at higher latitudes would be less resilient to phase shifting effects of light.

This seems to be confirmed by our finding of higher PRC amplitude and circadian light

sensitivity in the northern line compared to the southern line. Furthermore, N. vitripennis´

positive correlation of circadian light sensitivity and latitude follows the same trend as its

critical photoperiod for diapause induction (Paolucci et al. 2013). These conflicting findings

can be unified when considering that complex network oscillators do not obey simple oscillator

theory predicting decreased light sensitivity with increased oscillator strength. In addition,

neuronal photoreceptor circuits could change light sensitivity properties of the input pathway

to the oscillator without changing the properties of the oscillator itself. Alternatively, neuronal

oscillator network properties can also modify the sensitivity to light (Van der Leest et al. 2009;

Ramkisoensing et al. 2014; Buijink et al. 2016; Beersma et al. 2017).

In Nasonia, longer circadian free-running periods under continuous darkness were

observed at higher latitudes (northern females = 26.3 h (SD ± 1.6 h), northern males = 24.8 h

(SD ± 1.0 h), southern females = 25.0 h (SD ± 0.3 h), southern males = 24.2 h (SD ± 0.3 h);

Chapter 2). This latitudinal cline in circadian period may have emerged due to the involvement

of the circadian system in diapause regulation, leading to earlier onset of autumn diapause at

higher (colder) latitudes (Hut and Beersma 2011; Hut et al. 2013). Although, the involvement

of the circadian system in diapause regulation seems established for Nasonia (Saunders 1974),

it might be species dependent and is therefore still under debate (Bradshaw and Holzapfel 2010;

Hut and Beersma 2011; Koštál 2011; Saunders and Bertossa 2011; Dolezel 2015). In Nasonia,

the longer free-running periods, deviating more from 24-h, require stronger Zeitgebers or

increased light sensitivity in order to maintain entrainment. Variation in free-running period

might be related to polymorphisms found for the per gene as described by Paolucci et al., 2016.

Further, light sensitivity might be adaptively increased to facilitate entrainment of more

deviating circadian periods, especially at environments closer to the Arctic where absolute light

intensities decrease. The more prominent phase advances in our PRCs support the hypothesis

that the longer circadian periods are compensated by increased PRC amplitude. To confirm our

explanation that a latitudinal cline in per-gene polymorphism may be responsible for the

increased circadian light sensitivity, further knowledge on the light resetting mechanism in

Nasonia is needed.

Light reception of the circadian system in Drosophila occurs mainly through the photo-

sensitive protein Cryptochrome, d-CRY (Emery et al. 1998, 2000b; Stanewsky et al. 1998). The

Nasonia homolog is photo-insensitive and shows more homologies with the mammalian type

Cryptochrome, m-CRY (Bertossa et al. 2014). In flies, light activated d-CRY interacts with the

TIM protein, leading to its degradation and thereby destabilisation of PER. Both TIM and PER

are the core components of the transcriptional-translational feedback loop which generates a

~ 24 hour oscillations. Light induced d-CRY degradation of TIM and PER in long photoperiods

or constant light constantly degrade these core clock proteins and render the entire molecular

pacemaker arrhythmic(Ceriani et al. 1999; Koh et al. 2006; Peschel et al. 2009). In addition to

Chapter 3

56

the d-CRY-dependent light input pathway to the circadian system in flies, there is also an opsin

based, d-CRY-independent light input pathway to fly circadian system (Helfrich-Förster et al.

2001; Yoshii et al. 2015). There are marked differences between the light resetting mechanism

of Drosophila and Nasonia. The d-CRY system in Drosophila allows all body cells to respond

to circadian light resetting, however, it is rather unlikely that the Nasonia nv-CRY protein is

light sensitive and therefore another resetting mechanism of the pacemaker is probable. It is

much more likely that circadian light resetting in Nasonia is more mammal-like, using

specialized photoreceptors that specifically project to circadian pacemaker neurons. This would

suggest regulation of circadian light sensitivity in Nasonia at different neurobiological levels,

perhaps allowing for circadian light sensitivity to be regulated independently from circadian

amplitude.

In conclusion, we showed that the circadian system of Nasonia vitripennis from northern

Europe has higher light sensitivity than from southern Europe, suggesting a positive latitudinal

cline in circadian light sensitivity. We propose that adaption to northern environments caused

earlier diapause induction to survive harsher conditions. Adaptation is probably caused by

polymorphisms of the core clock gene period (Paolucci et al. 2016), leading to longer free-

running periods and longer critical photoperiods for diapause induction. As a result we suggest,

circadian light sensitivity had to increase to maintain normal natural entrainment. This study

shows that measuring latitudinal adaptation generates insight into various evolutionary

selection pressures acting on the circadian system.

Acknowledgment

We thank the INsecTIME consortium for their support. This work was supported by the Marie

Curie Initial Training Network programme INsecTIME (Grant number PITN-GA-2012-

316790).

university of groningen latitudinal differences in the

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