RESEARCH ARTICLE

Plausible link between circa‘bi’dian activity rhythms and circadianclock systems in the large black chafer Holotrichia parallelaYuta Kawasaki1, Hitoshi Nishimura1 and Sakiko Shiga1,2,*

ABSTRACTTwo-day rhythms, referred to as circa‘bi’dian rhythms, have beenreported in humans and mosquitos. However, these rhythms onlyappear under constant conditions, and the functional mechanisms of2-day rhythms were unknown. Here, we report clear circabidianrhythms of large black chafers (Holotrichia parallela, Coleoptera:Scarabaeidae) in both the laboratory and field. Under 12 h:12 h light:dark (L:D) conditions at 25°C, H. parallela appeared on the ground atthe beginning of the dark phase every 2 days. Under constantdarkness, H. parallela exhibited free-running with a period of 47.9±0.3 h, suggesting the existence of a clear circabidian rhythmentrained to two 12 h:12 h L:D cycles. Phase responses of thecircabidian rhythm to light pulses occurred under constant darknessin a phase-dependent manner. Phase responses suggest that thereare two circadian cycles, each consisting of a less-responsive andmore-responsive period, in a circabidian oscillation, and thecircabidian rhythm is driven by the circadian clock. A mark–recapture study showed that beetles repeatedly appeared on thesame tree approximately every 2 days. However, the periodicity wasnot as rigid as that observed under laboratory conditions in thatindividuals often switched appearance days. For instance, a largeprecipitation made the 2-day rhythm shift phase by half a cycle of therhythm at a time. We propose a novel function of the circadian clockcharacterized by the release of an output signal every two cycles toproduce the 2-day rhythm.

KEY WORDS: Two-day rhythm, Pheromone trap, Phase–responsecurve, Beetle, Mark–recapture

INTRODUCTIONVarious environmental factors that are associated with physical andbiological features change periodically on the Earth, and organismshave evolved endogenous rhythms with periods that approximateenvironmental cycles. Many organisms have circadian rhythmsdriven by the circadian clock with a period of about a day (Dunlapet al., 1999). The suppression of activities during an appropriatetime of a day by the circadian clock is an adaptive behaviorassociated with escape from predation and other environmentaldangers (DeCoursey et al., 1997, 2000). Significant deviationfrom cyclical periods of activity for 24 h decreases bothsurvivorship and reproduction rates (Spoelstra et al., 2016).

Endogenous rhythms with periods similar to other environmentalcycles such as tidal, lunar and yearly cycles have also beenprogressively studied (Numata and Helm, 2014; Kaiser et al.,2016). Therefore, the use of an endogenous rhythm with a cyclethat corresponds to environmental changes probably allowsorganisms to appropriately anticipate and prepare theirphysiological states.

However, rhythmicity associated with cycles that inconsistentlyapproximate cyclical environmental changes has been reported.Two-day rhythms, referred to as circa‘bi’dian rhythms, wererecorded in humans and mosquitos. For instance, Kleitman (1963)successfully developed 48 h oral temperature rhythms by enforcing48 h sleep–wakefulness routines in humans. In an undergroundbunker, ∼50 h activity cycles were observed in an isolated malesubject exposed to constant light conditions, but the cyclical periodof the body temperature rhythmwas ∼25 h (Aschoff et al., 1967). Inthe mosquito Culiseta incidens, a ∼46 h flight rhythm was observedin constant dark conditions (Clopton, 1984, 1985). However,occurrences of circabidian rhythms in these two species wereinfrequent and observed only when organisms were kept for longperiods under constant conditions without time cues, or when theywere forced to entrain to 2-day cycles (Czeisler et al., 1980;Clopton, 1985). Therefore, it is not known whether circabidianrhythms actually occur in nature.

In the large black chafer Holotrichia parallela (Coleoptera:Scarabaeidae; previously called Lachnosterna morosa), a seriousagriculture pest in East Asia, adult populations appear on the groundevery 2 days (Yoshioka and Yamasaki, 1983). Measurements ofpheromone titers in the pheromone glands of field-collected femalesshowed that a peak occurred every 2 days (Leal et al., 1993).However, this periodicity has only been described in groups, andindividual periodicity and the mechanisms behind the rhythm arenot known. In contrast to humans and mosquitos, H. parallelapopulations seem to have 2-day periodicity in the field. If the 2-dayperiodicity of this species is formed by an endogenous free-runningcircabidian rhythm under constant conditions, H. parallelarepresents a good model that could aid our understanding of howan enigmatic rhythm with a period different from an environmentalcycle is assembled by a clock mechanism and how it actually worksin the field.

In this study, we chronobiologically examined the individualground emergence rhythms in field-collected H. parallela, andfound that this species has a very stable circabidian rhythm underconstant darkness. Furthermore, the rhythm was perfectly entrainedto every two cycles of 12 h light and 12 h darkness. We alsoexamined phase responses to light pulses, which suggested that thecircabidian rhythm is determined by a circadian clock. Finally, weperformed mark–recapture studies, which demonstrated that the2-day periodicity was not as rigid as that observed under solitarylaboratory conditions, and the beetles switched the day ofappearance in the field. The circadian clock-driven mechanismsReceived 22 May 2017; Accepted 3 September 2017

1Department of Biology andGeosciences, Graduate School of Science, Osaka CityUniversity, Sumiyoshi, Osaka 558-8585, Japan. 2Department of Biological Science,Graduate School of Science, Osaka University, Machikaneyama, Toyonaka 560-0043, Japan.

*Author for correspondence (shigask@bio.sci.osaka-u.ac.jp)

S.S., 0000-0002-9507-0532

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might contribute to a half-cycle temporal shift of the circabidianrhythm.

MATERIALS AND METHODSInsectsAdult large black chafers, H. parallela (Motschulsky 1854), werecollected on the riverbed of the Yamato River (34°35′14″N, 135°30′16″E) in July of 2012 (Fig. 1) and the riverbed of the Yodo River(34°43′41″N, 135°31′31″E) in June to August of 2012–2015(Figs 2 and 3).

Recording of activity rhythmThe beetles were individually kept in a polystyrene cylindricalcontainer (11.5 cm height, 7 cm diameter). Two-thirds of thecontainer was filled with culture soil (Sakata Seed Corporation,Yokohama City, Japan), and the side wall of each container wascovered with aluminium foil to prevent light exposure to beetlesunder the soil surface. Activity of the beetles on the ground wasindividually recorded under 12 h light and 12 h darkness (12 h:12 hL:D) for 10 days and subsequent constant darkness (DD) for10 days at 25±1°C. The light source was fluorescent light and theintensity was 1.75 W m−2. During the recording period, a leaf of theJapanese cherryPrunus yedoensis ‘Somei-yoshino’was provided asfood every 5 days or once at the beginning of the experiment, andapproximately 25 ml of water was sprayed on the soil surface whenit dried. The top of the container was covered with a transparentglass plate. A color image of the surface of the soil was taken every6 min with a web camera (DC-NCR13U, Hanwha Japan, Tokyo,Japan). According to differences in the total pixel values betweentwo serial photos, beetle movements were detected and plotted ona double-plotted actogram, and the free-running period wasdetermined using a chi-square periodogram (Enright, 1965;Sokolove and Bushell, 1978).

Total pixel values were calculated per photo, and the value of thedifference, w (x, y), was calculated as:

wðx; yÞ ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiXx

Xy

fðRnðx; yÞ � Rn�1ðx; yÞÞ2 þ ðGnðx; yÞ � Gn�1ðx; yÞÞ2s

þ ðBnðx; yÞ � Bn�1ðx; yÞÞ2g;ð1Þ

where (x, y) represents pixel coordinates, Rn(x,y) represents redvalues per pixel, Gn(x,y) represents green values per pixel andBn(x,y) represents blue values per pixel. We also calculated thethreshold value of w based on the amount of noise from all recordedphotos for each beetle. w values above threshold indicated that thebeetle had moved since the previous photo was taken, and a score of‘1’ was plotted for that time. w values below threshold were given ascore of ‘0’. Activity occurring over a 48 h period was plotted on adouble-plotted actogram.

To examine the biological clock cycle driving the circabidianrhythm, the phase responses of the rhythm to light pulses wereexamined under DD. We used light-transparent sponges instead ofsoil. Water was added to the sponge through a cotton thread from awater tank set below the container. A cherry leaf was given as food,and the leaf was replaced twice during the recording period in mostcases. In other cases, the leaf was only provided at the beginning ofthe recording period. Activity recordings were conducted for9–10 days under DD; a 3 h-light pulse was then provided twice in48 h at different phases of the circabidian time (CbT), and activitywas then recorded for 8–10 days under DD. The light intensitymeasured inside of the sponge pile was 0.626 W m−2.

An actogram was drawn as the soil activity recordings wereconducted. We set the activity onset as a phase reference point ofCbT=36. A computer programwas written to determine the onset ofthe activity phase as follows. (1) We first searched for the mostactive 18 h time zone over 48 h, starting at 10:00 h Japan standardtime (JST). Total activity scores were calculated, and ranged from 0to 180 for each 18 h time zone. The starting time of each 18 h timezone was delayed by 1 h, so the first zone started at 10:00 h JST andended at 18:00 h JST, the second one started at 11:00 h JST andended at 19:00 h JST, and so on. Time zones with the highest scoreswere selected. If two or more high-scoring zones were present, theearlier zone was selected. (2) In the high zone, the first appearanceof 15 sets of continuous activity was selected, and the starting time(JST) of the appearance was designated as the activity onset point.The activity onset was determined every 48 h starting at 10:00 hJST. (3) We then evaluated the activity onset point. There was amaximum of five activity onset points before and after the lightpulse. The median activity onset time before and after the lightpulses was calculated for each beetle, and activity onset times thatdiffered from the median by 6 h or more were regarded as outliers.In the 48 h cycle with outlier points, the first appearance of 15 sets ofcontinuous activity was searched again during a period of themedian ±6 h, and the starting time was set as the revised activityonset points. (4) To determine the CbT of the onset of activity, a linewith inclination of τ from the chi-square periodogram was drawn atthe position where the sum of the squared deviations of the activityonset point from the line was lowest. Based on τ and the position ofthe line, we calculated the CbT of the activity onset point, and thedifference between the onset of the activity phase in the periodbefore versus after the light pulse was calculated in CbT.

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Fig. 1. Activity rhythms of adult Holotrichia parallela on the ground. (A) Amale beetle and (B) a female beetle. Activity (black bars) recorded in two 48 hperiods is shown in the double-plotted actogram over 20 days. The beetleswere recorded under 12 h light and 12 h dark (12 h:12 h LD) conditions for10 days and under constant dark conditions for 10 days, as indicated by thewhite (light) and black (dark) horizontal bars above the actogram. The twographs to the right show a chi-square periodogram analysis during light anddark (LD, upper) and constant dark (DD, lower) conditions. Time on the graphshows the period of the rhythm analyzed by the chi-square periodogram.

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Study sitesField observations were conducted on the riverbed of Yamato Riverin Osaka (Fig. S1). Temperature in the field ranged from −2.4 to36.7°C in 2011 and −3.1 to 37.9°C in 2012.

Mark–recaptureThe mark–recapture study was conducted on tree E (Ulmusparvifolia, 2.7 m) in 2011, and trees A (U. parvifolia, 3.4 m) andE in 2012. The distance between trees A and E was 87 m (Fig. S1).We checked the presence of beetles on the trees from 3 May to 8September in 2011 and from 30 May to 26 September in 2012.During the observation period, the observer went to the trees atsunset every day with a lantern, and H. parallela aggregating on thetree were collected using an insect net and a stepladder. Anindividual three-digit number was engraved on the elytra of eachbeetle, and the number of newly captured (unmarked) beetles andmarked beetles was recorded separately every night. Beetlesaggregating on 10 trees, designated A–J in Fig. S1, were countedat sunset on 28–29 July 2011 with the help of five observers. Beetleson the tree were collected, and were immediately released after themarked number on the elytra was checked and the number ofunmarked beetles was counted.

Beetle collection with pheromone trapsThe sex pheromone of H. parallela, which has two components(major component, L-isoleucine methyl ester; minor component,linalool) was prepared (Leal et al., 1992). Ammonia was added to

L-isoleucine methyl ester hydrochloride (C7H15NO2·HCl; CAS no.18598-74-8, Wako Pure Chemical Industries, Ltd, Osaka, Japan) toobtain L-isoleucine methyl ester, and L-isoleucine methyl ester andlinalool (C10H18O; CAS no. 78-70-6, Nacalai Tesque, Inc., Kyoto,Japan) were mixed at a 4:1 ratio. The pheromone solution (100 µl)was placed in a 500 µl plastic tube, and a piece of filter paper wasused as the pheromone source. A 1.5 l plastic bottle was set up underthe pheromone tube as a non-lethal trap to collect male adults, andthe trap was hung on tree A. From 17 June to 8 October 2010, thenumber of H. parallela captured in the trap and the number ofbeetles aggregating on tree Awas examined every 8 days except for27–28 August. The pheromone for this trap was renewed at 12:00 hJST every day. The number of beetles was counted every hour from06:00 h JST until 06:00 h JST of the following day, and collectedbeetles in the trap were immediately released after counting wascompleted. Most beetles were alive, and the number of beetlesaggregated on tree A was counted by eye and categorized into thefollowing three groups: −, n=0; +, n=1–9; ++, n=10 or more.

Statistical testsMann–Whitney U-tests, t-tests (one-sided test), chi-square tests, chi-square goodness of fit tests and two-way ANOVAwere conducted byusing R software (Ihaka and Gentleman, 1996; http://cran.r-project.org) with an additional package twoway.anova (http://aoki2.si.gunma-u.ac.jp/R/src/twoway_anova.R). For detection of rhythmicity, chi-square periodogram analysis was performed (P<0.05), and aperiod with the peak value of the variance in the analysis was

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Fig. 2. Activity rhythms ofH. parallela underconstant dark conditions with light pulses.Activity (black bars) recorded over two 48 hperiods is shown in the double-plottedactogram over 24 days. Gray bars indicate aperiod with no data and yellow bars indicate alight pulse. Red dashed lines indicate theapproximate onset of a phase of activity. Lightpulses were emitted at circabidian times(CbT)=4.0 h (A), CbT=34.5 h (B), CbT=47.0 h(C), CbT=17.5 h (D), CbT=39.5 h (E),CbT=22.5 h (F) and CbT=8.0 h (G). Somebeetles exhibited circadian-like activity afterthe light pulses, as seen in G.

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determined as the rhythm period (Enright, 1965; Sokolove andBushell, 1978).

RESULTSActivity rhythmUnder 12 h:12 h LD conditions, male and female beetles appearedon the ground at the beginning of the dark phase every 2 days(Fig. 1). We occasionally observed that the beetles appeared justbelow the soil surface and remained there until the light was turnedoff. After the light was turned off, the beetles then emerged onto thesoil, and were observed feeding and walking. The duration on theground was 6.3±1.6 h (mean±s.d., n=10), and the beetles mostlystayed underground for the rest of the day and all of the followingday. In the container, the beetles usually stayed about 5 cm belowthe soil surface during the rest phase. Even under DD, theiremergence rhythm continued, and the free-running period was 47.9±0.3 h (n=7) in males and 47.6±0.3 h (n=3) in females. Holotrichiaparallela exhibited a clear endogenous rhythm with a period ofapproximately 48 h under DD, and it entrained to two cycles of12 h:12 h LD (Fig. 1).

Phase responses of the circabidian rhythm to light pulsesBecause the free-running period before the light pulse did not differsignificantly between females (47.6±0.3 h, n=28) and males (47.8±0.5 h, n=36, t-test, P>0.05), we plotted the phase shift values offemales and males together (Fig. 2). In 61 of the 65 beetles (oneindividual unsexed), the circabidian rhythm continued after the lightpulses, and the free-running period was 47.7±0.4 before the lightpulse and 47.7±0.6 h afterwards (paired-sample t-test, P>0.05,n=61). However, the onset of the activity phase was advanced,delayed or unchanged depending on the pulse phase (Fig. 2A–F).We calculated each phase shift value in CbT and plotted the shiftvalue for the phase at which the light pulse was given (Fig. 3A). TheCbT is a time scale covering one full circabidian period (∼48 h)during an oscillation under DD, and we set the onset of the activityphase at 36 h in CbT. To examine the dependency of shift values oncircabidian phases, we divided a 48 h cycle in CbT into eightperiods (t1–t8) of 6 h each to compare phase shift values betweentwo consecutive periods (Fig. 3A). Significant differences weredetected between the phase shift values of t3 (median=−1.6 h) and t4(median=1.25 h, Mann–Whitney U-test, P=0.004), between t6(median=0.4 h) and t7 (median=−2.6 h, U-test, P=0.002), andbetween t7 and t8 (median=−0.4 h, U-test, P=0.046). At thebeginning of t8, it appeared that the phase shift direction changedfrom delayed to advanced. In contrast, shift values were small, and asignificant difference was not observed between t1 (median=0.3 h)and t2 (median=−1.9 h, U-test, P=0.112), between t2 and t3 (U-test,P=1.000), between t4 and t5 (median=0.4 h, U-test, P=0.262), andbetween t5 and t6 (median=0.4 h, U-test, P=0.800). If the circadianclock cycles twice in a 48 h CbT, a change of phase shift valueshypothetically occurs between t3 and t4, and between t7 and t8. Ourdata agree with this. Less-responsive periods to light theoreticallyoccur at t1–2 and t5–6, and significant differences were not detected inthese periods. The lack of a significant difference between t2 and t3and between t4 and t5 suggests that phase shift values in t3–4 areweaker than those in t7–8 because during t3–4, H. parallela do notreceive light under soil and their sensitivity might be weakened.When phase shift values in the first and second 24 h CbT periodwere superimposed, phase shift direction appeared similarbetween the first and second cycles (Fig. 3B). When the 24 htime frame of the superimposed plot was divided into fourperiods (T1–T4), significant differences were not detectedbetween T1 (median=0.2 h) and T2 (median=−0.7 h, U-test,P=0.413), but were detected between T2 and T3 (median=−1.8 h,U-test, P=0.006), and between T3 and T4 (median=0.4 h, U-test,P=0.002).

Circadian-like activity rhythms were observed after the lightpulse in four of the 65 beetles (Fig. 2G, Fig. S2). This suggests thatH. parallela exhibits an oscillator with a cycle of about 24 h.Because the circadian-like rhythm was observed in response to lightpulses given at a variety of phases (5.0, 8.0, 15.5, or 43.0 h in CbT),no specific phase seemed to change from a circabidian to a circadianrhythm.

Appearance times in the fieldTo understand the emergence times in the field, we counted thenumber of male H. parallela collected using a pheromone trap seton tree A (Chinese elm Ulmus parvifolia) every hour for 24 h(Fig. S1). Male beetles were mostly trapped within a few hours ofsunset from June to October (Fig. 4). A large number ofH. parallelawere trapped from 25–26 June to 12–13 August, and beetlescontinued to appear until 7–8 October. In addition to the trappedmales, male and female H. parallela appeared on the tree on which

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Fig. 3. Phase responses of H. parallela to 3 h light pulses emitted atdifferent circabidian times under constant darkness. Advanced anddelayed phase shift values are plotted as positive and negative values on theordinate. The onset of activity is set as a phase reference point of CbT=36 h.(A) One oscillation of a circabidian cycle divided into eight terms (6 h each)from t1 to t8 to examine the dependency of shift values on circabidian phases(see Results). Cycles of less-responsive and more-responsive phases occurtwice in a circabidian oscillation. Gray dots indicate individuals provided withless food compared with individuals denoted by black dots, but activity was notaffected. (B) The same data as in A, with the first half (CbT=0–24 h, red) andlast half (CbT=24–48 h, blue) of a circabidian cycle superimposed on a 24 htime frame (N=61), divided into four periods (T1–4).

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the trap was set. These beetles stayed on the tree throughout thenight, and mating was often observed within 1 h of sunset.Holotrichia parallela stayed on the tree throughout the night, andtheir appearance and disappearance were mostly synchronized withsunset and sunrise, respectively (Fig. 4).

Individual appearance rhythms on the treeTo examine the individual rhythms in the field, we conducted amark–recapture study on tree E in 2011 and on trees A and E in 2012(Fig. S1). The first appearance dates of H. parallela were 3 June in2011 and 4 June in 2012. We set 3 June as a reference date, andnamed it the ‘first’ day in both 2011 and 2012. Days of emergencewere distinguished between odd days and even days. The 1st (3June), 3rd (5 June), 5th (7 June), 7th (9 June), etc., days weredesignated as ‘odd days’ and the 2nd (4 June), 4th (6 June), 6th (8June), 8th (10 June), etc., days were the ‘even days’ for both years.

Beetles were marked at the first appearance. Beetles marked on anodd day were categorized into the ‘odd day’ group, and thosemarked on an even day were placed in the ‘even day’ group.

In 2011 on tree E, the number of beetles in the even and odd daygroup did not differ significantly (Table 1). The rate of recapture ofbeetles on the marked tree two times or more was 26.1%, and thenumber of recaptured beetles was 37. Twenty-four of 37 beetlesappeared only on an even day, counted from the first appearance (themarked day), during the observation period. For example, malespecimen no. 67 appeared 2, 4, 8, 10, 14, 16, 18 and 22 days afterthe marked day (Fig. 5). The other 13 beetles reappeared on oddnumbered days, counted from the first appearance. Femalespecimen no. 2 reappeared on day 2, 4, 6, 8, 9, 13, 15, 17 andadditional days after the first appearance (Fig. 5). We named thelatter behavior ‘temporal switching’, and the occurrence of temporalswitching between the odd day and even day groups did not differ

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Fig. 4. Appearance time of H. parallelaon tree A (Ulmus parvifolia). Columnsshow the number of male H. parallelacaptured by the pheromone trap. Symbolsrepresent the number of beetles on thetree: ++, 10 or more; +, fewer than 10; −,0. Downward arrows show the time ofsunset and upward arrows show the timeof sunrise. Abscissa indicates time of dayfrom 06:00 h to 06:00 h.

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significantly in 2011 (Table 2). Of the 13 beetles, nine, three andone switched appearance once, twice and four times, respectively(Fig. S3). The last day of appearance in 2011 was 1 September.In 2012, the last appearance day was 19 September on trees A and

E. More beetles were found on the larger tree A than on tree E, butthe rate of recapture of beetles on the tree two times or more did notdiffer significantly between trees A and E (Table 1, chi-square test,χ2=3.396E–30, d.f.=1, n=459, P>0.05). In contrast to the 2011population, the population size in 2012 was significantly larger inthe odd day group than in the even day group on tree E, but it was notdifferent on tree A (Table 1 and Fig. 6). In 2012, 33 beetles from twotrees exhibited temporal switching, and 29, 3 and 1 beetles switchedappearance days once, twice and three times, respectively. Forbeetles marked on trees A and E, the temporal switching rate wassignificantly larger in the even day group (Table 2). Most beetleswere observed repeatedly only on the tree where the beetle wasmarked, but some reappeared on different trees. For instance, femalespecimen no. 330 first appeared on tree A, but it appeared on tree Ethe second time and tree A at a later time (Fig. 5). This phenomenonwas referred to as spatial switching. The spatial switching rate wasabout 10%, and it did not differ significantly between beetlesmarked on trees A and E (Table 2; Fig. S3).Fig. 6 shows seasonal variation in the emergence of beetles. We

counted the number of male and female beetles separately each day,and a significant difference in the number of beetles was detectedbetween days but not between sexes (two-way ANOVA, P>0.05 forsex, P<0.01 for day; Table S1). In 2011 at tree E, the odd day andeven day groups mostly reappeared on different days in June andJuly. However, in August and September, when very few beetlesappeared, synchronization of appearance days occurred between thetwo groups (Fig. 6A). In 2012, differences in population size wereobserved between the odd day and even day groups on tree E

(Table 1). Two-day periodic appearances were only obvious in thelarge population of the odd day group (Fig. 6B). On tree A, an abruptdisappearance of unmarked (newly captured) beetles occurred in theeven day group after 20 June (Fig. 6B). There was heavy rain fromTyphoon No. 4 and a tropical cyclone that occurred on 19–22 June(Japan Meteorological Agency 2012; http://www.data.jma.go.jp/fcd/yoho/data/typhoon/T1204.pdf). The heavy rains led to the floodingof the ground at the observation site on 22 June (an even day), and nobeetles appeared on that day on trees A and E (see inset in Fig. 6B).The beetles appeared again on 23 June. After this submersion, beetlesfrom the even day group exhibited temporal switching (appearing onthe odd days), and complete synchronization occurred for 9 days from23 June to 1 July between the odd day and even day groups on bothtrees, with the exception of one specimen on 28 June on tree E(Fig. 6B). This explains the high percentage of temporal switching inthe even day group in 2012 (Table 2). After the heavy rain, morebeetles were newly captured on the odd days than on the even days,suggesting that many of the unmarked beetles also exhibited temporalswitching after the submersion.

Distribution range of H. parallelaBecause the percentage of recaptured beetles on trees A and E wasnot very high, we suspected that the beetles might be appearing onneighboring trees. We examined the occurrence of beetles markedon tree E and unmarked beetles on 10 trees (including tree E) on 28and 29 July 2011 (Fig. S1; Fig. 7). The total number of appearancesfor 2 days on the 10 trees was 112, but no beetles were captured attrees B, F and J. Beetles originally marked on tree E were mostlyfound on tree E (n=14). Regarding other trees, only a single markedbeetle was found on trees A (sex, unknown) and G (female), but allother beetles were unmarked.

We chased seven beetles that flew away from tree A around sunrisein June and July of 2012, and found that they dug into the soil in anarea of 15 m diameter around tree A (Fig. 8). These observationssuggested that most H. parallela individuals repeatedly visited thesame tree that was close to their daytime resting place.

DISCUSSIONEndogenous 2-day rhythm in H. parallelaThe present study revealed that H. parallela exhibited clearendogenous circabidian rhythms with regard to its appearance onthe ground under laboratory conditions. Individual appearances inthe field also fitted an approximate 2-day cycle, but individualsoften switched appearance days, presumably based onenvironmental conditions.

10 20 300

0 10 20 30

40

40

Days after 1st appearance

34396799

12925

577592

14210228240310

116677

330395

Male

Female

Male

Female

2011

2012

Fig. 5. Representative individual plots of H. parallelaappearance in the field. The horizontal axis indicates thenumber of days from the first appearance. Orange cells andblue cells indicate an even and odd numbers of days,respectively, counted from the first appearance. The green cellhighlighted for female specimen no. 330 in 2012 indicatesspatial switching: no. 330 was marked on tree A, appeared attree E 12 days after the first appearance (green cell), and wasback on tree A on subsequent days.

Table 1. Number of marked Holotrichia parallela and recapture rate onUlmus parvifolia trees (A and E)

Year-tree N

No. marked

PRecapturerate (%)

Odd daygroup

Even daygroup

2011-E 142 80 62 0.131 26.12012-A 322 167 155 0.378 22.72012-E 137 112 25 3.05E−12 22.6

The table shows the year and tree on which H. parallela were marked and thetotal number (N ) of beetles. P-values are the results of chi-square goodness offit tests between odd and even day groups. The recapture rate indicates thepercentage of beetles recaptured two or more times on the marked tree.

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Circabidian rhythms were observed as ∼50 h periodicity ofsleep–wakefulness in humans under conditions free of time cues(Czeisler et al., 1980) and ∼46 h of flight activity rhythms in themosquito C. incidens under constant conditions (Clopton, 1984).These circabidian rhythms were labile and subsequently returned tocircadian or became obscure. Because circabidian rhythms inhuman subjects are associated with sleep–wakefulness activitiesobserved during desynchronization from body temperature rhythmsthat oscillate in a circadian manner, researchers concluded thatcircabidian rhythms resulted from the uncoupling of multi-oscillators under constant conditions (Aschoff et al., 1967).Clopton (1984) basically followed this idea to interpret therhythms of mosquitos. In these reports, the circabidian rhythmwas thought to be expressed only under non-natural conditions asone unique character of the coupled circadian clock system.However, the circabidian rhythm observed in H. parallela clearlydiffered from labile rhythms. In H. parallela, the 2-day rhythmappeared stable under both LD and DD experimental conditions,and the rhythm was also observed in the field. Their circabidianappearance or activity on the ground might contribute to a reductionin prey risk and conservation of energy for reproduction. A lowerappearance level seems disadvantageous because the opportunity tofind a mate is diminished by half. Holotrichia parallela might copewith a reduction in mating opportunities by increasing matingefficiency. In fact, the mating period in H. parallela is shortcompared with that of other congeneric species (Matsumoto, 2010).They might increase mating frequency to copulate with morepartners than other congeneric species.

Entrainment of circabidian rhythm to light and dark cyclesThe circabidian rhythm in H. parallela entrained to two cycles of12 h:12 h LD, with activity every other night. Because beetles stayunderground during the day, they are barely exposed to light.Beetles with a free-running period shorter than 48 h probably cameup to the surface of the ground before the lights were turned off, thusfollowing an internal clock. Actually, we occasionally observedmovement of the ground surface before the lights were turned off onthe appearance day, suggesting that beetles came up just below (noton) the soil surface to sample the light. On non-appearance days,light-sampling behavior was not observed. Light-sampling behaviorhas been shown in some cavern-dwelling bats and the flying squirrelGlaucomys volans (Twente, 1955; DeCoursey, 1986), which leavesthe den after arousal to sample light through the sampling porthole.If light was seen, the squirrel returned to the den to take a short napbefore venturing out again (DeCoursey, 1986). When the light wasoff during sampling, the squirrel left the den. This unusualentrainment process is similar to that in H. parallela.Some H. parallela individuals displayed a free-running period

longer than 48 h, which allowed them to come up to the surface ofthe ground after the light was off and to sample light at dawn toentrain LD cycles. In the field, the beetles actually stayed on the tree

throughout the night until dawn (Fig. 4). Under laboratoryconditions, H. parallela only stayed on the ground for 6 h afterthe light was turned off. We think that this resulted from confinedsolitary conditions without a mate and the inability to fly (Fig. 1).

Holotrichia parallela probably monitor light intensity todetermine the timing of emergence on the ground. In a congenericspecies, Holotrichia loochooana, adult emergence and matingbehaviors were observed in the laboratory under natural lightconditions. Emergence on the ground and female calling behaviorsoccurred at a certain range of light intensity (Kawamura et al.,2001). As H. parallela mating time is limited (Yoshioka andYamasaki, 1983), it seems important for H. parallela individuals towait for the start of darkness just below the surface of the ground andto appear quickly on the ground to facilitate populationsynchronization.

Biological clocks driving the circabidian rhythmTo determine the cycle of the clock that drives the circabidianrhythm, we examined the phase responses of the rhythm to thezeitgeber light. Researchers have demonstrated that phase advancesor delays in rhythm in response to a zeitgeber depend on clockphases, and this is a unique characteristic of oscillator-type clocks(Pittendrigh, 1960). Phase-response curves have been constructedfor different kinds of biological rhythms, and curve periods ofapproximately 24 h, 12.4 h and 1 year have been revealed incircadian, circatidal and circannual rhythms, respectively(Pittendrigh and Minis, 1964; Akiyama, 1997; Miyazaki et al.,2005; Satoh et al., 2008). In the circadian clock, clock phases aredivided into the subjective day and night periods. In the cricketGryllus bimaculatus, circadian activity rhythms exhibit littleresponse to light pulses during the subjective day, but a delay inthe first half and an advance in the last half of the subjective nightwere observed (Okada et al., 1991). In H. parallela, we observedtwo sets of the less-responsive period and more-responsive (delay oradvance) period in one circabidian cycle. This result suggests that acircabidian cycle is composed of two cycles of the circadianoscillator. However, phase responses were too obscure to draw a finecurve. To obtain clear phase-response curves a greater number ofbeetles might be necessary. Meanwhile, four beetles showedcircadian-like activity rhythms after light pulses, suggesting thatH. parallela are capable of driving activity in a circadian manner.Phase responses to light pulses by the circabidian rhythm andchange of the circabidian rhythm to a circadian-like activity rhythmsupport the idea that the circadian clock generates the circabidianrhythm in H. parallela.

Clopton (1984) proposed a mechanism for the circabidian rhythmin mosquitos, based on a coupled oscillator model. When mosquitosor humans are kept free from timing cues, oscillator couplingbetween circadian clocks becomes weakened, and a labile oscillatorproduces a 2-day rhythm that is uncoupled from the rigid 24 hoscillator. The uncoupled labile oscillator lengthens its period and

Table 2. Occurrence of temporal switching and spatial switching in H. parallela

Year-tree Nrecapture

% Temporal switching (N)

P*

Spatial switching

Odd day group Even day group % P‡

2011-E 37 22.2 (18) 47.3 (19) 0.109 – –

2012-A 73 12.5 (40) 72.7 (33) 1.66 E−07 13.7 0.5712012-E 31 0 (26) 80.0 (5) 1.02 E−06 9.7

The table shows the year and tree on which H. parallela were marked and the number (Nrecapture) of beetles that reappeared two or more times on the markedtree. P-values are the results of chi-square tests on temporal switching rates between odd and even day groups (*) and spatial switching between tree A andE in 2012 (‡).

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0

No.

of i

ndiv

idua

lsN

o. o

f ind

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Jul. Sep.Jun. Aug.

A

0

20

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Jul. Sep.Jun. Aug.

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21 22 230

5

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E, odd E, even A, odd A, even

22 230

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21 22 23Jun. Jun. Jun. Jun.

No.

of

indi

vidu

als

No data

No data

E, odd

E, even

E, odd

E, even

A, odd

A, even

Fig. 6. Seasonal changes in the emergence of H. parallela on the riverbed of Yamato River. Data from (A) 2011, (B) 2012. The beetles were separatelyplotted on the graph depending on whether they were marked on odd days or even days (on trees A and E) and males and females are plotted together(see Results for details). The first day was set as 3 June in both 2011 and 2012. Beetles marked on the 1st (3 June), 3rd (5 June), 5th (7 June), 7th (9 June), etc.,were put in the odd day group, and thosemarked on the 2nd (4 June), 4th (6 June), 6th (8 June), 8th (10 June), etc., were put in the even day group. Black columnsshow recaptured marked individuals and gray columns show newly captured individuals. The data from 21 to 23 June 2012 (indicated by the red box), whentemporal switching mainly occurred, are extracted in the inset below. See Table 1 for sample size.

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recouples to a rigid 24 h oscillator when its period double. Anothermechanism for creating a longer rhythm than intrinsic oscillatorshas been hypothesized as beats (Bünning, 1962). Beats originatefrom a difference in period length between oscillators that are notcoupled. Semi-lunar periodic phenomena may originate from thecooperation of a daily (about 24 h) and a tidal rhythm (about12.4 h). However, we think clock mechanisms of H. parallelarhythms are different from oscillator coupling or beats, because theappearance of the circabidian rhythm is rigid, not labile, both underLD and constant conditions, and no oscillator components otherthan the 24 h oscillator were detected in phase-response

experiments. Further rhythmic components with different cyclesproducing a beat every 48 h are inconceivable.

We suggest the most parsimonious interpretation is that thecircabidian output is produced every two oscillations of thecircadian clock entraining with 24 h light–dark cycles. Thefrequency demultiplication hypothesis, in which biologicalrhythms with a long period are derived from rhythm with a shortperiod through a process of frequency demultiplication, has beensupported in the circasemilunar rhythm in Pontomya oceana(Soong and Chang, 2012). The results indicate the presence of thecounter-mechanisms in which cycle numbers of the circadianoscillations are counted to make circasemilunar emergence rhythm.To make the circabidian rhythm, two circadian cycles might also becounted in H. parallela.

Spoelstra et al. (2016) suggested that biological clocks withcycles that do not coincide with environmental cycles are eliminatedby natural selection. With regard to biological clocks, the evolutionof a cycle that is not associated with an environmental cycle may bedifficult. If there are adaptive values associated with a 2-day rhythm,it might be easy to produce circabidian rhythms by modifying theoutput systems of the circadian clock, which releases a signal everytwo cycles of the clock.

Circabidian rhythms in the fieldThe 2-day periodicity of individuals was observed in the field.However, its periodicity was not constant, and the absence of aperiod was often observed. As beetles marked on tree Ewere seldomfound on other trees (Fig. 7), we think that H. parallela individualsrarely go to other trees, but they frequented bushes around the treeon which they were marked. We also observed them eating Poaceaegrasses.

In the field,H. parallela occasionally showed temporal switchingof the 2-day rhythm. The period 22–23 June 2012 represented a timewhen beetles were not able to come out on the ground because ofheavy precipitation, which resulted in the synchronization ofappearances in the odd day and even day groups. For thissynchronization, the even day group changed to appear on odddays. In 2012, the majority of beetles started to appear on tree E andwere marked from late June after the heavy precipitation, thus,

0

10

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28 Jul. 29 Jul 28 Jul. 29 Jul. 28 Jul. 29 Jul.

0

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28 Jul. 29 Jul. 28 Jul. 29 Jul. 28 Jul. 29 Jul. 28 Jul. 29 Jul.

0

10

20

28 Jul. 29 Jul. 28 Jul. 29 Jul.

(N=43)

(N=0) (N=6) (N=22)

(N=16) (N=0) (N=9) (N=12)

(N=4) (N=0)

No.

of b

eetle

s 28 Jul. 29 Jul.

A

B C D

E F G H

I J

Fig. 7. Number of H. parallela individuals that appeared on trees A–J on28–29 July 2011.White columns show the number of beetlesmarked on tree Eand black columns indicated unmarked beetles. Onemarked beetle was foundon trees A and G, and most were on tree E. No beetles were found on treesB, F and J.

10 m

Yamato River

15 m

21 Jun.

6 Jul.

24 Jul.

25 Jun.

B×

×A

Fig. 8. Flight range of H. parallela atsunrise. The cross indicates trees A andB. Colored circles indicate the point wherethe beetle went underground from treeA. Circles of the same color indicatebeetles observed on the same day. Thedashed line shows the area of a circle of15 m diameter around tree A.

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resulting in population size differences between odd and even daygroups. Such a clear synchronization was not found in 2011,although there was heavy rain in September. Emergencesynchronization seems to depend on environmental changes, andrain is only one factor.In 2011, some even day beetles showed temporal switching that

resulted in a synchronized occurrence with the odd day group inAugust and September when population size was smaller. It isknown that H. loochooana and Dasylepida ishigakiensis, whichbelong to the sameMelolonthinae subfamily asH. parallela, exhibitcalling and mating behaviors that occur in the population at a fixedperiod of the day (Kawamura et al., 2001; Arakaki et al., 2004;Yasui et al., 2007; Fukaya et al., 2009; Tokuda et al., 2010).Yoshioka and Yamasaki (1983) reported that H. parallela matesoon after sunset, and we also confirmed this in the field. Maleswere caught by pheromone traps within a few hours of sunset,indicating that males quickly fly toward pheromones, and matingsubsequently occurs. If copulation occurs during a restricted time ofday in these species, sympatric and synchronized populationoccurrences are needed to increase copulation efficiency.Temporal switching in H. parallela may play a role in thesynchronization of sympatric populations. Considering the factthat individual activities recorded in the laboratory never switchedto the next day, temporal switching was probably induced byenvironmental and social cues. Holotrichia parallela usescircabidian rhythms in a flexible manner to change emergencedays in order to adjust to the changing environment.How does the temporal switch occur? If the circabidian rhythm

wasmade by a hypothetical circabidian clock, a phase shift of a half-cycle (∼24 h) of the clock had to occur to switch the appearancedays. For an oscillator-type clock, it is hard to make a half-periodphase shift at one time (Benstaali et al., 2001), and it usually takes atransient period to complete a full shift. If temporal switching wasadaptive for the beetle (e.g. facilitated avoidance of aversiveconditions or increased population size), the beetles had to developsome mechanism to make a shift of appearance occur one day at atime. The circabidian rhythm driven by the circadian clock systemmight facilitate an immediate switch in appearance day. Circabidianoutput might be activated or suppressed after counting two circadianoscillations. If so, some unknown environmental stimulus maymodulate the day-counting system to give an output after one orthree circadian oscillations, thus resulting in temporal switching.Here, we propose a novel functional circabidian rhythm that isaffected by the circadian clock. In future experiments, we aim toclarify the molecular and neuronal bases of the involvement of thecircadian clock in the circabidian rhythm.

AcknowledgementsWe are grateful to Dr Takeshi Matsumoto of the Japanese Society ofScarabaeoidology for his advice on the field study. We would also like to thankDr Tetsuro Shinada and Dr Tetsuo Tsujioka at Osaka City University and Dr YasuoMukai at Local Organization for Promotion of a Japanese Rural Area ‘Nitta’ for theirtechnical advice on pheromone synthesis, image processing analysis and mark–recapturemethod. This work was supported by aGrant-in Aid for Scientific Research(Houga) 23657060 provided by the Japan Society for Promotion of Science to S.S.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: S.S.; Methodology: Y.K., H.N., S.S.; Software: Y.K.;Validation: Y.K., S.S.; Formal analysis: Y.K., H.N.; Investigation: Y.K., H.N.; Datacuration: Y.K., H.N., S.S.; Writing - original draft: Y.K., H.N.; Writing - review &editing: Y.K., S.S.; Supervision: S.S.; Project administration: S.S.; Fundingacquisition: S.S.

FundingThis work was supported by a Grant-in-Aid for Scientific Research (Houga)23657060 provided by the Japan Society for Promotion of Science to S.S.

Supplementary informationSupplementary information available online athttp://jeb.biologists.org/lookup/doi/10.1242/jeb.163253.supplemental

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