Pleiotropic effect, clock genes, and reproductive isolation

 The mechanism by which a clock gene pleiotropically controlling life history and behavioral traits causes reproductive isolation is explained using a model species, the melon fly, Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae). Melon flies mate once a day, at dusk. The population selected for life history traits exhibits correlated responses in the time of mating during the day. For example, the fly populations selected for faster (slower) development have an earlier (later) time of mating. A circadian rhythm controls the time of mating. The circadian periods in constant darkness were about 22 h in lines selected for a short developmental period and about 31 h in lines selected for a long developmental period. The data on crosses between the selected lines indicated that the developmental period is controlled by a polygene, whereas the circadian period may be controlled by a single clock gene. These results suggest a clock gene pleiotropically controls developmental and circadian periods in the melon fly. Reproductive isolation may often evolve as an indirect (pleiotropic) consequence of adaptation to different environments or habitats. For example, niches that are temporally or seasonally offset can select organisms with different developmental characteristics. These developmental differences can inadvertently cause reproductive isolation by a variety of means including shifts in mating activity patterns. The difference in time of mating between populations selected for developmental period translated into significant prezygotic isolation, as measured by mate choice tests. If the mating time between populations differed more than 1 h, the isolation index was significantly higher than zero. These findings indicate that premating isolation can be established by a pleiotropic effect of a clock gene. There are many examples in which the difference in timing of reproduction prevents gene flow between populations, such as the egg spawning time in marine organisms, the flowering time in angiosperms, and the time of mating in insects. In such organisms, if genetic correlations between circadian rhythm and reproductive traits exist, multifarious divergent selection for life history traits would often accelerate the evolution of reproductive isolation through clock genes. Natural populations may diverge in reproduction time through drift, direct natural selection for time of reproduction, or as a by-product effect of genetic correlations. In any case, clock genes are keys in reproductive isolation. Received: January 31, 2002 / Accepted: July 29, 2002 Acknowledgments I am grateful to Tetsuo Arai, Akira Matsumoto, Takashi Matsuyama, Toru Shimizu, Aya Takahashi, Teiichi Tanimura, Tetsuya Toyosato, and Yasuhiko Watari for useful discussion, and to the responsible editor and two anonymous reviewers for helpful suggestions. I also thank Yoshihiko Chiba, Norio Ishida, Emi Koyama, Kazuhiko Sakai, and Takaomi Sakai for useful information. My work on speciation has been supported by a Grant-in-Aid for Scientific Research (KAKENHI 14340244) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.     

Male courtship song in circadian rhythm mutants of Bactrocera cucurbitae (Tephritidae: Diptera)

Pulse train intervals (PTI) of courtship song were differentiated between circadian clock mutants of the melon fly, Bactrocera cucurbitae (Coquillett) (Tephritidae: Diptera). We analysed the male mating song of B. cucurbitae flies of two mutant strains that differed in circadian locomotor rhythm by a LabVIEW programming system. Flies with a short circadian rhythm (S-strain) had shorter PTI than those with a long circadian rhythm (L-strain) in the two age groups tested, young and old. Young flies showed longer PTI than old flies, but no interaction between strain and age was found in PTI. There was a significant interaction between strain and age for pulse train duration (PTD), whereas no stable difference was found in PTD between S- and L-strains. These results suggest a positive correlation between the length of the circadian locomotor rhythm and PTI of courtship song sounds in B. cucurbitae.     

Circadian rhythm and time of mating in Bactrocera cucurbitae (Diptera: Tephritidae) selected for age at reproduction

Chrono-biological traits were changed by selecting for life-history traits via a genetic linkage controlling both time-related behavioural and life-history traits. Behavioural traits were compared between lines selected for young (Y-lines) and old (O-lines) age at reproduction in the melon fly, Bactrocera cucurbitae (Coquillett). Adults from O-lines, which survive longer than flies from Y-lines, mated later in the day and had a longer period of circadian rhythm in the locomotor activity than those from Y-lines. Flies from F(1) reciprocal crosses had an intermediate time of mating and periods of circadian rhythm between that of the parents, indicating a genetic basis to these traits. The presence of these behavioural differences across the selection lines indicates that chrono-biological traits exhibit correlated responses to selection on age at reproduction. The correlated responses in the behavioural traits to selection for life-history traits are discussed from two points of view: pleiotropy and inadvertent selection.     

GENETIC CORRELATIONS BETWEEN LIFE-HISTORY AND BEHAVIORAL TRAITS CAN CAUSE REPRODUCTIVE ISOLATION

Reproductive isolation may often evolve as an indirect (pleiotropic) consequence of populations adapting to different environments or habitats. For example, niches that are temporally or seasonally offset can select for organisms with different developmental characteristics. These developmental differences can inadvertently cause reproductive isolation by a variety of means including shifts in mating activity patterns. Here, we show a genetic correlation between a life-history trait (developmental period) and a behavioral trait (time of mating) that causes significant premating isolation in the melon fly, Bactrocera cucurbitae (Diptera: Tephritidae). Fly lines selected for short and long developmental periods differ in their preferred times of mating during the evening. This difference translates into significant prezygotic isolation, as measured by mate choice tests. If the time of mating between two populations differed more than one hour, the isolation index was significantly higher than zero. These indicate that premating isolation can be established if the developmental period is divergently selected for. If such genetic correlations are ubiquitous in many organisms, multifarious divergent selection for life-history traits would often accelerate the evolution of reproductive isolation. We speculate that reproductive isolation may have been evolved via genetic correlations among time-related traits, for example, developmental period and time of mating, as in other organisms.     

Time, love and species

Physiological and behavioral phenomena of many animals are restricted to certain times of the day. Many organisms show daily rhythms in their mating. The daily fluctuation in mating activity of a few insects is controlled by an endogenous clock. The fruitfly, Drosophila, is the most suitable material to characterize the genetic basis of circadian rhythms of mating because some mutants with defective core oscillator mechanism, feedback loops, have been isolated. D. melanogaster wild-type display a robust circadian rhythm in the mating activity, and the rhythms are abolished in period or timeless null mutant flies (per(01) and tim(01)), the rhythms are generated by females but not males. Disconnected (disco) mutants which have a severe defect in the optic lobe and are missing lateral neurons show arrhythmicity in mating activities. Thus, the lateral neurons seem to be essential for the circadian rhythm in mating activity of Drosophila. Furthermore, an anti-phasic relation in circadian rhythms of the mating activity was detected between D. melanogaster and their sibling species D. simulans. The Queensland fruit flies or wild gypsy moth also show species-specific mating rhythm, suggesting that species-specific circadian rhythms in mating activity of insect appear to cause a reproductive isolation.     

Circadian rhythmicity within single cells of Paramecium bursaria

Cell populations of Paramecium bursaria show mating reactivity in the light period, but not in the dark period, when exposed to a light-dark cycle (LD 12:12). After they are transferred to constant-light (LL) conditions (1,000 lux), they continue to show a circadian rhythm of mating reactivity. The rhythm gradually dampens in LL so that mating reactivity in populations becomes arrhythmic in LL within 2 weeks. We wanted to know whether the arrhythmicity of this population was due to the absence of circadian rhythmicity within each individual cell, or merely due to asynchrony of a population of individually rhythmic cells. Therefore, single cells were isolated randomly from an arrhythmic population that had been in LL for a long time. Then the mating reactivity of these single cells was individually tested every 3 hr for 2 days. Each single cell showed a circadian mating rhythm in LL. This shows that the disappearance of the mating rhythm in cell populations under LL is not caused by disappearance of circadian rhythmicity within individual cells, but is due to desynchronization among cells in a population. When an arrhythmic population in LL is darkened for 9 hr, the mating reactivity rhythm of the cell population reappears. This occurs by resynchronization of the rhythms among individual cells, as can be shown by exposing single cells to pulses of 9 hr of darkness. This dark treatment causes phase shifts of single-cell rhythms, and a phase response curve is obtained for this stimulus. This phase-shifting behavior explains the efficacy of 9-hr dark pulses in restoring the population's rhythm.     

Time-Dependent Effects of Trichloroethylene on Motor Activity in Rats

Circadian variations in acute and subacute neurobehavioural effects of trichloroethylene (TRI: 1.2 g/kg i.p.) were investigated in the rat under a light: dark = 12:12 hr cycle. An acute effect of TRI evaluated by decreased muscle tone was maximal during the early dark phase (21:00). A subacute effect of TRI was evaluated by a continuous recording of spontaneous locomotor activity in the rat. The circadian rhythm in spontaneous locomotor activity was extensively impaired by the injection of TRI for three consecutive days. Spectral analysis of spontaneous locomotor activity showed that ultradian periods became more dominant than the circadian period, and the 1//fluctuation of the spectrum disappeared after the injection of TRI. The effect of TRI on the circadian rhythm in spontaneous locomotor activity was circadian-phase dependent, and the treatment of TRI at 09:00 provoked greater circadian rhythm impairment than that at 21:00. The mechanisms of the time-dependent effect of TRI on neurobehaviour are the subject of further investigation.     

Time-Dependent Effects of Trichloroethylene on Motor Activity in Rats

Circadian variations in acute and subacute neurobehavioural effects of trichloroethylene (TRI: 1.2 g/kg i.p.) were investigated in the rat under a light: dark = 12:12 hr cycle. An acute effect of TRI evaluated by decreased muscle tone was maximal during the early dark phase (21:00). A subacute effect of TRI was evaluated by a continuous recording of spontaneous locomotor activity in the rat. The circadian rhythm in spontaneous locomotor activity was extensively impaired by the injection of TRI for three consecutive days. Spectral analysis of spontaneous locomotor activity showed that ultradian periods became more dominant than the circadian period, and the 1//fluctuation of the spectrum disappeared after the injection of TRI. The effect of TRI on the circadian rhythm in spontaneous locomotor activity was circadian-phase dependent, and the treatment of TRI at 09:00 provoked greater circadian rhythm impairment than that at 21:00. The mechanisms of the time-dependent effect of TRI on neurobehaviour are the subject of further investigation.     

Circadian regulation of egg-laying behavior in fruit flies Drosophila melanogaster

Significant progress has been made in our understanding of the neurogenetics of circadian clocks in fruit flies Drosophila melanogaster. Several pacemaker neurons and clock genes have now been identified and their roles in the cellular and molecular clockwork established. Some recent findings suggest that the basic architecture of the clock is multi-oscillatory; the clock mechanisms in the ventral lateral neurons (LN(v)s) of the fly brain govern locomotor activity and adult emergence rhythms, while the peripheral oscillators located in antennal cells regulate olfactory rhythm. Among circadian phenomena exhibited by Drosophila, the egg-laying rhythm is unique in many ways: (i) this rhythm persists under constant light (LL), while locomotor activity and adult emergence become arrhythmic, (ii) its circadian periodicity is much longer than 24h, and (iii) while egg-laying is rhythmic under constant darkness, the expression of two core clock genes period (per) and timeless (tim), is non-oscillatory in the ovaries. In this paper, we review our current knowledge of the circadian regulation of egg-laying behavior in Drosophila, and provide some possible explanations for its self-sustained nature. We conclude by discussing the existing limitations in our understanding of the regulatory mechanisms and propose few approaches to address them.     

Population genetic structure of the melon fly, Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae) based on mitochondrial cytochrome oxidase (COI) gene sequences

Population genetic structure of melon fly analysed with mitochondrial cytochrome oxidase I gene suggested that melon fly populations across the globe is homogeneous with non-significant variation of 0.000-0.003 base substitutions per site. Test isolates representing various geographic situations across the world were placed in 26 mitochondrial haplotypes based on variations associated with a maximum of three mutational steps and the predominant haplotype i.e. H1 was present in all melon fly populations except Hawaiian population. Evolution of mtCOI gene suggested that the fly could have originated some 0.4 million years ago. The present study also indicated that the B. cucurbitae population expansion is an event of post Pleistocene warm climatic conditions with small number of founder population. The invasion of B. cucurbitae in Hawaii was associated with the large population size and the global presence of the fly is associated with human mediated dispersal. The very low genetic variation suggested that the fly management might be possible by large scale sterile insect techniques programme.     

Correlated changes in circadian clocks in response to selection for faster pre-adult development in fruit flies Drosophila melanogaster

Although, circadian clocks are believed to be involved in the regulation of life-history traits such as pre-adult development time and lifespan in fruit flies Drosophila melanogaster, there is very little unequivocal evidence either to support or refute this. Here we report the results of a long-term study aimed at examining the role of circadian clocks in the temporal regulation of pre-adult development in D. melanogaster. We employed laboratory selection protocol for faster pre-adult development on four large, outbred, random mating populations of Drosophila. We assayed pre-adult development time and circadian period of locomotor activity rhythm of these flies at regular intervals of 5–10 generations. After 50 generations of selection, the overall egg-to-adult duration in the selected stocks was reduced by ~29 h (~12.5 %) relative to controls, with the selected populations showing a concurrent reduction in time taken to hatching, pupation and wing pigmentation, by ~2, ~16, and ~25.2 h, respectively. Furthermore, selected populations showed a concomitant reduction in the circadian period of locomotor activity rhythm, implying that circadian clocks and development time are correlated. Thus, our study provides the first ever unequivocal evidence for the evolution of circadian clocks as a correlated response to selection for faster pre-adult development, suggesting that circadian clocks and development are linked in fruit flies D. melanogaster.     

Circadian rhythms in the mating behavior of the cockroach, Leucophaea maderae

Mating behavior of small populations of virgin males and females of the cockroach Leucophaea maderae were continuously monitored via time-lapse video recording in controlled laboratory conditions. The time of onset of copulation was found to be rhythmic in a light cycle of 12 h light alternated with 12 h of darkness, with the peak of mating behavior occurring near the light to dark transition. This rhythm persisted in constant dim red illumination and constant temperature. In constant conditions, the period of the rhythm was slightly less than 24 h, with a peak of copulation during the late subjective day. These data demonstrated that mating behavior is gated by a circadian clock. When males and females were taken from light cycles that were 12 h out of phase, a bimodal rhythm was observed with one peak in the males' late subjective day and a second peak of equal amplitude in the late subjective day of females. The results indicated that circadian systems in both males and females contribute to the circadian rhythm in copulation. Bilateral section of the optic tracts (OTX) of both males and females abolished the rhythm, but the rhythm persisted when OTX females were paired with intact males or when OTX males were paired with intact females. Furthermore, when OTX males or OTX females were paired with intact animals that were 12 h out of phase, a bimodal rhythm was still observed. These results suggested that the circadian pacemaker in the optic lobes of both male and female cockroaches participates in the control of mating, but that a pacemaker outside the optic lobes is also likely involved. Finally, it was shown that the female's olfactory response (measured by electroantennogram) to components of the male sex pheromone exhibited a circadian rhythm, but the data suggested the peripheral olfactory rhythm is not likely to be involved in the rhythm of mating behavior.     

The effects of pinealectomy and blinding on the circadian locomotor activity rhythm in the Japanese newt,Cynops pyrrhogaster

We examined the effects of pinealectomy and blinding (bilateral ocular enucleation) on the circadian locomotor activity rhythm in the Japanese newt, Cynops pyrrhogaster. The pinealectomized newts were entrained to a light-dark cycle of 12 h light and 12 h darkness. After transfer to constant darkness they showed residual rhythmicity for at least several days which was gradually disrupted in prolonged constant darkness. Blinded newts were also entrained to a 12 h light/12 h dark cycle. In subsequent constant darkness they showed free-running rhythms of locomotor activity. However, the freerunning periods noticeably increased compared with those observed in the previous period of constant darkness before blinding. In blinded newts entrained to the light/dark cycle the activity rhythms were gradually disrupted after pinealectomy even in the presence of the light/dark cycle. These results suggest that both the pineal and the eyes are involved in the newt's circadian system, and also suggest that the pineal of the newt acts as an extraretinal photoreceptor which mediates the entrainment of the locomotor activity rhythm.Abbreviations circadian period - DD constant darkness - LD cycle, light-dark cycle - LD 12:12 light-dark cycle of 12 h light and 12 h darkness     

Unequal coupling between locomotor pacemakers of the German cockroach,Blattella germanica (L.)

Male adult German cockroaches, Blattella germanica (L.), expressed robust locomotor circadian rhythmicity under 28 degrees C and constant darkness (DD) conditions. By surgically severing the connections between the optic lobes and midbrain and their subsequent regeneration, we demonstrated that the locomotor circadian pacemaker was located in the optic lobes and that it controlled the locomotor circadian rhythm through neural pathways. From the results that unilaterally optic tract severed males still showed locomotor circadian rhythmicity (93.1%, n=29) without significantly changing the circadian period (tau) or level of motor activity, we concluded that the right and left optic lobes each contain a circadian pacemaker competent to drive the locomotor circadian rhythm. These two pacemakers were strongly coupled since only one rhythm was expressed when the pacemakers were independently exposed to opposite lighting conditions (DD or LL) at the same time. However, an unequal contribution of each pacemaker in determining the overt circadian period was found under constant dim light (10 lux) conditions, revealing a major-minor coupling relationship between these two pacemakers, so that the unilaterally blinded male expressed either a LL-rhythm with a circadian period of 24.27+/-0.21 h (41.7%, n=24) or a DD-rhythm with a circadian period of 23.43+/-0.19 h (58.3%, n=24). However, higher intensity of photic information (200-300 lux) could overpower this relationship and cause the minor pacemaker to lead the rhythm.     

Characteristics of the circadian activity rhythm in common marmosets (Callithrix j. jacchus)

The circadian activity rhythm of the common marmoset, Callithrix j. jacchus was investigated by long-term recording of the locomotor activity of 15 individuals (5 males, 10 females) from 1.5 to 8 years old, both under constant illumination and under LD 12:12. The mean period of the spontaneous circadian rhythm was 23.2 ± 0.3 h. Neither sex-specific differences nor a systematic influence of light intensity on the spontaneous period were observed, but the period was dependent on the duration of the trial and on the age of the individual. Due to the short spontaneous period, in LD 12:12 there was a distinct advance of the activity phase with respect to the light time and a masking of the true onset of activity by the inhibitory direct effect of low light intensity during the dark time. After an 8 h delay shift of the LD 12:12, re-entrainment of the circadian activity rhythm required an average of 6.8 ± 0.7 days; the average re-entrainment time after an 8 h phase advance of the LD cycle was 8.6 ± 1.3 day. This directional effect is ascribed to characteristics of the phase-response curve. No ultradian components were observed, either in the LD-entrained or the free-running circadian activity rhythm.     

Neural pathways involved in mutual interactions between optic lobe circadian pacemakers in the cricketGryllus bimaculatus

The circadian locomotor rhythm of the cricketGryllus bimaculatus is primarily generated by a pair of optic lobe circadian pacemakers. The two pacemakers mutually interact to keep a stable temporal structure in the locomotor activity. The interaction has two principal effects on the activity rhythm, i.e., phase-dependent modulation of the freerunning period and phase-dependent suppression of activity driven by the partner pacemaker. Both effects were mediated by neural pathways, since they were immediately abolished after the optic stalk connecting the optic medulla to the lobula was unilaterally severed. The neural pathways were examined by recording locomotor activity, under a 13 h light to 13 h dark cycle, after the optic nerves were unilaterally severed and the contralateral optic stalk was partially destroyed near the lobula. When the dorsal half of the optic stalk was severed, locomotor rhythm mostly split into two components: one was readily entrained to the given light-dark cycle and the other freeran with a marked fluctuation in freerunning period, where the period of the freerunning component was lengthened or shortened when the onset of the entrained component occurred during its subjective night or day, respectively. The phase-dependent modulation of activity was also observed in both components. However, severance of the ventral half of the optic stalk resulted in appearance only of the freerunning component; neither the phase-dependent modulation of its freerunning period nor the change in activity level was observed. These results suggest that neurons driving the mutual interaction and the overt activity rhythm run in the ventral half of the proximal optic stalk that includes axons of large medulla neurons projecting to the cerebral lobe and the contralateral medulla.Abbreviations LD light dark cycle - freerunning period     

Circadian mating activity and effect of pheromone pre-exposure on pheromone response rhythms in the moth Spodoptera littoralis

Mating in moths is generally mediated by female-produced sex pheromones. Mating activity, female pheromone production/release and male pheromone responsiveness all show diurnal variations in many species. We found that the response of the male Egyptian cotton leafworm, Spodoptera littoralis, to sex pheromone gland extracts showed a diel rhythm in olfactometer tests, and the variation was persistent for at least 1 day in constant darkness. High male response to sex pheromone was correlated in time with high mating and locomotor activity. Male S. littoralis, maintained in constant darkness and exposed to pheromone gland extracts on a daily basis, showed an induced temporal variation in response after several days, in contrast to unexposed males. This suggests that in the absence of other external zeitgebers, exposure to sex pheromone may function to synchronise circadian behavioural rhythms in male moths. The daily rhythm in mating activity in S. littoralis is also shown to be persistent for at least 2 days in constant darkness. Pairs mated significantly less when either the male or female had been raised in a light:dark cycle 10 h out of phase, indicating that the proposed circadian rhythm in mating activity is composed of rhythmic mating preference/ability in both sexes.     

Circadian rhythm of locomotor activity in the Japanese honeybee, Apis cerana japonica

Abstract.  To reveal circadian characteristics and entrainment mechanisms in the Japanese honeybee Apis cerana japonica , the locomotor-activity rhythm of foragers is investigated under programmed light and temperature conditions. After entrainment to an LD 12 : 12 h photoperiodic regime, free-running rhythms are released in constant dark (DD) or light (LL) conditions with different free-running periods. Under the LD 12 : 12 h regime, activity offset occurs approximately 0.4 h after lights-off transition, assigned to circadian time (Ct) 12.4 h. The phase of activity onset, peak and offset, and activity duration depends on the photoperiodic regimes. The circadian rhythm can be entrained to a 24-h period by exposure to submultiple cycles of LD 6 : 6 h, as if the locomotive rhythm is entrained to LD 18 : 6 h. Phase shifts of delay and advance are observed when perturbing single light pulses are presented during free-running under DD conditions. Temperature compensation of the free-running period is demonstrated under DD and LL conditions. Steady-state entrainment of the locomotor rhythm is achieved with square-wave temperature cycles of 10 °C amplitude, but a 5 °C amplitude fails to entrain.     

Circadian rhythms of locomotor activity and temperature selection in sleepy lizards, <Emphasis Type="Italic">Tiliqua rugosa</Emphasis>

This study examined whether the daily rhythms of locomotor activity and behavioural thermoregulation that have previously been observed in Australian sleepy lizards (Tiliqua rugosa) under field conditions are true circadian rhythms that persist in constant darkness (DD) and whether these rhythms show similar characteristics. Lizards held on laboratory thermal gradients in the Australian spring under the prevailing 12-hour light : dark (LD) cycle for 14 days displayed robust daily rhythms of behavioural thermoregulation and locomotor activity. In the 13-day period of DD that followed LD, most lizards exhibited free-running circadian rhythms of locomotor activity and behavioural thermoregulation. The predominant activity pattern displayed in LD was unimodal and this was retained in DD. While mean levels of skin temperature and locomotor activity were found to decrease from LD to DD, activity duration remained unchanged. The present results demonstrate for the first time that this species’ daily rhythm of locomotor activity is an endogenous circadian rhythm. Our results also demonstrate a close correlation between the circadian activity and thermoregulatory rhythms in this species indicating that the two rhythms are controlled by the same master oscillator(s). Future examination of seasonal aspects of these rhythms, may, however, cause this hypothesis to be modified.