Effects of body clock disruption in submarines and other environments with non-24-hour days—from research to use

Most organisms living on this planet possess circadian clocks that orchestrate their daily physiological and behavioral rhythms to the 24-h rotation period of the earth [1]. However, under special conditions in which the cycling periods do not extend over 24 h, the circadian systems may be challenged. In the laboratory, manual non-24-h conditions are established to study the basic features of circadian rhythms, although such conditions are very rare in nature. However, in human societies, a substantial group of people lives or works under non-24-h schedules. The non-24-h conditions were observed to lead to a decrease in adaptability to the environment in all tested organisms across different kingdoms, including bacteria, fungi, plants, and animals.

The circadian clocks are well conserved across species; thus, we will first introduce the knowledge derived from circadian studies in model organisms and the next focus on summarizing studies in humans. The former part would help to elucidate circadian misalignment in circadian rhythms, metabolism, sleep and the cognitive and behavioral consequences.

Circadian clocks govern life processes at multiple layers, including the molecular, cellular, organism and integral levels. At the organism level, circadian clocks are hierarchically composed of master clocks located in the pacemakers and peripheral clocks located in other tissues. In mammals, the pacemaker is the suprachiasmatic nucleus (SCN) in the anterior of the hypothalamus. In birds and reptiles, the eyes, pineal gland, or SCN are circadian pacemakers, depending on the species [1–3]. The peripheral clocks have their own circadian rhythms, but they can also be synchronized by pacemakers through the nervous and endocrine pathways [4].

Although circadian rhythms are endogenous, they are subject to the influence of environmental, genetic, physiological and pathological factors (Fig. 1) [11, 12]. It has been demonstrated that disruptions in circadian rhythms impair adaptability in a variety of organisms [13]. In humans, compromised circadian rhythms cause chronic health consequences, including an increased risk of gastrointestinal illness, loss of bone mineral density, coronary artery disease, endocrine disruption, metabolic syndrome, and cancer. Moreover, disrupted circadian rhythms also affect emotions, cognition and performance (Fig. 1) [14–18].

The cycling periods of the environment, such as light and temperature, are defined as T-cycles. In a certain range, circadian rhythms can be adjusted to match the environmental periods, a process that is termed entrainment or synchronization. Circadian rhythms are both robust and flexible. Within a certain range, T-cycles that deviate from the natural 24-h cycles can also induce rhythms with the same periodicity as the environment. In this scenario, the endogenous circadian periodicity may be masked by the non-24-h T-cycles. The factors that can entrain circadian rhythms are known as zeitgebers, synchronizers or time givers [2, 19].

To experimentally determine the entrainment ranges, organisms are exposed to a series of fixed T-cycles or gradually changing T-cycles [20]. In addition, the entrainment range to an environmental cue can be inferred by the phase-response curve (PRC) information of the species. Given that according to the PRC of a species, the maximum phase delay and maximum phase advance are X hours and Y hours, respectively, the entrainment range of this species to the indicated cue would range from (24 h – Y) to (24 h + X) hours [1]. PRC represents the nonparametric effects of zeitgebers on entrainment. However, parametric components are also involved in determining the entrainment range. The parametric model assumes that light changes the velocity of the circadian rhythms for entrainment, while the nonparametric model emphasizes the phase shift [21].

Circadian rhythms run free when the T cycles are beyond the entrainment ranges. Under LD 9:9, squirrel monkeys (Cebidae Saimiri) display a core body temperature (CBT) rhythm with a period of ~ 24 h instead of 18 h, suggesting the occurrence of a nearly free-running CBT rhythm (Fig. 2c) [26]. An approach has been proposed to measure the free-running period based on the displayed periods under non-24-h cycles beyond the entrainment ranges [28, 29]. Importantly, the rhythms beyond the entrainment ranges should be called superposition (or superimposition) [1, 24, 25]. Superposition may result in a period different from the one measured in a constant condition [20].

Despite the different entrainment ranges, different organisms exhibit rhythms with endogenous periods after transfer to constant conditions [24], suggesting the endogenous characteristics of the sustainability of circadian rhythms. In a study, the pregnant mice were kept in LD 28 or LD 20, and their progenies were also raised in these T-cycles until they reached puberty. The progenies showed free-running periods soon after they were placed in constant conditions [30]. The sole recorded exception was that hydrodictyon maintains a period of ~ 17.5 h in the constant dark for at least 3 days after having been retrieved from the LD 10.5:7 condition [24].

Different physiological and behavioral rhythms may also have different entrainment ranges within one species or even within individuals [20], suggesting the existence of independent oscillators. In addition to affecting the pacemaker of the circadian clock, zeitgebers also bypass it, directly impacting the overt rhythms, which causes internal desynchronization between different physiological processes as a consequence when T cycles are out of the entrainment ranges, e.g., the sleep-wake cycles and the CBT.

The molecular mechanisms determining the entrainment range have not been elucidated. The only clue regarding this mechanism was that in mice, Clock mutants exhibit a wider entrainment range (LD 24 to LD 32) compared to wild-type mice, suggesting that circadian clock genes play critical roles in regulating entrainment [31]. Mutants with less robust rhythms may be more prone to environmental entrainment, although such passive rhythms have no anticipating property, unlike circadian rhythms.

All the available evidence suggests that living under circumstances with cycling periods beyond the entrainment ranges is harmful to reproductive fitness. Cyanobacteria strains possessing different endogenous circadian periods were mixed, and after a month in culture, the strain with a period resonating with the environment prevailed, while the strain with a period different from the environment failed in the competition [33, 34, 38]. Several models have been proposed to explain the clock-controlled fitness in Cyanobacteria, including the limited resource model, diffusible factor model cell-to-cell communication model, and the escape-from-light hypothesis [44], which may also help to characterize the enhanced fitness by the circadian clock in other organisms [37, 45]. The escape-from-light hypothesis postulates that in the early stage of evolution, the circadian clock acts to segregate light-sensitive reactions temporally to nighttime, e.g., cell division and DNA damage repair, to avoid the deleterious effects of radiation and oxidation from sunlight [37, 45].

Growth and flowing time were extensively affected, although the results varied in different species. A comparison of the growth of tomato under different LD conditions demonstrated that tomato grows most quickly in LD 12:12 but very slowly in either LD 24:24 or LD 6:6 [36]. Similar experiments have been conducted in a variety of species, including house sparrow, jack bean (Canavalia ensiormis), N. crassa, and squirrel monkey (Saimiri sciureus), which indicate that light regimens with periods that deviated dramatically from 24 h hampered growth or survival [2, 26, 46]. In a previous study, several plants were tested in a series of LD cycles, ranging from LD 5 s:5 s to LD 12:12; for instance, soybean (Soja max (L.) IMper) under LD 1 min:1 min showed a markedly chlorotic phenotype and a notable reduction in size, contained numerous spots of dead tissue, and showed a tendency to die prematurely [47]. Of course, the day length also contributes to the comprehensive influences on plants.

It has been demonstrated that in many organisms, non-24-h cycles have extensive negative or deleterious effects on survival and competitive fitness. Lesion of the SCN in antelope ground squirrels (Ammospermophilus leucurus) led to the loss of circadian rhythmicity in locomotor activity and an increased ratio of loss caused by predation [48]. The tau mutation in the enzyme casein kinase 1ε (CK1ε) had a dramatic influence on circadian periods in mice, with the heterozygous mice showing a two-hour shorter period, while the homozygous mice showed a four-hour shorter period. In the semi-natural environment, the relative frequency of the tau allele decreased to 20% from the initial half in 14 months due to disadvantages in both adult survival and the recruitment of juveniles into the cohorts [49].

In addition to fitness, misalignment of circadian rhythms with non-24-h cycling conditions impairs many physiological and metabolic processes. Moreover, the disruption of circadian rhythms leads to abnormalities in cognition in animals. The circadian control of cognition and behavior has been demonstrated in many animal species. Disruption of mouse circadian rhythms by LD cycles led to extensive ramifications in metabolism, brain function and behavior [50]. For instance, in LD 10:10, mice exhibited decreased cognitive flexibility and changes in emotionality, which were consistent with changes in neural architecture, including shorter dendritic length and reduced complexity of neurons in the medial prefrontal cortex [51].

In addition, non-24-h cycles shorten life spans in many tested organisms. For instance, Pittendrigh and Minis raised Drosophila under the conditions of T = 21 h and T = 27 h, and the life spans of Drosophila in both conditions were considerably shorter than those lived in T = 24 h [39].

A hypothesis proposed that when the cycling zeitgeber period is close to a demultiplicative period of the free-running period, the oscillator may also be entrained, which is known as frequency demultiplication [52]. However, frequency demultiplication is more likely to represent a passive response to cycling environmental stimuli than circadian oscillations [2, 24, 36, 52, 53].

Circadian disruption profoundly affects health and performance in military and commercial vessel crews. During a 90-day voyage, which is the longest voyage recorded to date, disturbance in circadian rhythms was reported in many Chinese crew members. Most of the crew members experienced sleep disorders, flagging, dysphoria and faintness. Some crew members attempted to overcome these conditions by chewing dry peppers or daubing menthol camphorate on their temples [95]. In maritime missions, circadian misalignment causes serious consequences, including high personnel turnover (30–50%) per voyage and requirements to train the new entrant [26].

However, the question remains of how we can overcome circadian misalignment and avoid deleterious consequences. The entrainment range needs to be considered. If the environmental period is within or close to the entrainment range, it may be useful to use countermeasures to adjust the circadian rhythms. If the environmental period is far beyond the entrainment range, it may be useful to construct a man-made 24-h-period microenvironment. It is also possible to combine both approaches. Some efforts have been made to lessen the deleterious effects of disrupted circadian rhythms and to improve health and performance, which primarily involved optimization of the watch schedules and modifications of the zeitgebers.

Optimized watch schedules may improve circadian rhythms and performance. Duplessis et al. [59] compared the impacts of different watch schedules (6-h on/8-h off and a compressed close-6 h watch schedule), and the wrist activity results showed that sleep duration was longer under the 6-h on/8-h off schedule. Sleep discontinuity appears to be considerably less severe on the 6-h on/12-h off watch schedule than that of the 4-h on/8-h off watch schedule. In contrast, the 6-h on/12-h off schedule allowed more continuous sleep [60], which might be explained by the fact that T12 is shorter than T18 compared to human FRP. The best approach is to use a 24-h roster instead of a non-24-h roster. A 24-h schedule was used during an operational tour broad the USS MARYLAND, a U.S. Navy ballistic missile submarine, and positive feedback regarding fatigue, energy level, sleep inertia, and morale were obtained from the crew [58]. In 2014, the US Navy ordered all submarines to change to a 24-h schedule by December 2014 [96, 97]. It is believed that the 24-h schedule works better to improve mood and prompt social interaction. However, to this end, the prerequisite will be that the work environments have sufficient accommodability for alternate teams.

Even under the same regimens, the adaptation varies among shift sections. Under 4-h on/8-h off schedules, the three watch groups showed different cognitive patterns, which suggests different degrees of adaptation [93]. The interval between shifts is also a determinant of the performance [98]. If shifts change too frequently, the circadian rhythms of the subjects will lose synchrony, similar to the effects of jet lag. If shifts change less often, some of the teams will work at a ‘low’ status, similar to the night workers. In a long-term voyage, inner cues, such as lights and mealtime, might act as weak daily cycling zeitgebers in the cabin without exposure to natural light. In this scenario, there are always some personnel working at artificial nighttime.

Individual variation in the flexibility of circadian rhythms has been reported in several studies [62, 65]. In fact, screening and employing personnel who show higher tolerance to non-24-h cycles or shifts has been suggested. However, although some people show adaptation in sleep/wake cycles [65], extensive modulations of other rhythms have been observed; thus, the potential desynchronization in their inner rhythms and its long-term impacts on health and performance need to be further evaluated.

Some commonly used medications, e.g., dextroamphetamine, caffeine and modafinil, can temporally preserve or restore alertness and vigilance as pharmacological countermeasures under sleep deficiency [99]. Some other hypnotics, e.g., nonbenzodiazepines and melatonin receptor agonists, can improve the quantity and quality of sleep with fewer side effects [100]. However, these medications are usually used on short-term missions instead of long-term missions [101]. Cognition-enhancing equipment, such as transcranial direct current stimulation (tDCS), is widely used, but there is little evidence supporting the anticipated effects of this equipment [102].

The strength of the zeitgeber, such as the light intensity, also affects the range of entrainment [20]. Blue or white light has been used for light therapy of circadian rhythm-associated disorders and enhancement of alertness and performance [103]. In the new 24-h watch schedule in submarines, highly correlated color temperature (CCT = 13,500 K) fluorescent light sources function better in promoting alertness and performance to be aligned with the environmental cues [97]. Light therapy can improve circadian rhythmicity and performance in short-term missions, which may result in further aggravation under long-term non-24-h schedules.

It has been demonstrated that exposure to gradually changing environmental cycles may enlarge the entrainment range, e.g., gradually increasing/decreasing LD periods and feeding periods [20]. LD twilight exposure led to wider entrainment ranges in hamsters or deer mice compared to rectangular LD cycles [21, 104]. Similarly, sinusoidal light-intensity cycles also broadened the entrainment limits in rats [105, 106]. The effects of enhancing the entrainment ranges by changing zeitgebers may be attributed to parametric mechanisms [21].

Overall, the influences of non-24-h environments on physiology and behavior warrant further study, both in basic research and applied research. Many factors need to be considered to optimize the performance. Similar non-24-h schedules are also widely implemented in maritime operations and industries in addition to submarines, including oil mining, merchant vessels and some other industries and air crews [93, 107], suggesting a substantial population size of affected subjects. In the future, space exploration will expose humans to non-24-h cycling environments. For orbital space crafts, the LD cycling period is approximately 90 min [108]. The self-rotation periods of the Moon and Mars are ~ 14 days and 24.65 h, respectively [109]. Some countries, including the US and China, are planning to send humans to the moon and Mars in the near future, demonstrating the importance of the adaptation of circadian rhythms in exotic environments.