Amplitude of Circadian Rhythms Becomes Weaker in the North, But
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bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272070; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Amplitude of circadian rhythms becomes weaker in the north, 2 but there is no cline in the period of rhythm in a beetle 3 4 Masato S. Abe1 , Kentarou Matsumura2 , Taishi Yoshii3 , Takahisa Miyatake2 5 1 Center for Advanced Intelligence Project, RIKEN, Tokyo, Japan 6 2 Graduate School of Environmental and Life Science, Okayama University, 7 Okayama, Japan 8 3 Graduate School of Natural Science and Technology, Okayama University, 9 Okayama, Japan 10 11 Corresponding author: 12 Takahisa Miyatake E-mail: [email protected] 13 Telephone number: +81-86-251-8339 14 Postal address: Evol Ecol Lab, Graduate School of Environmental and Life Science, 15 1-1-1 Tsushima-naka, Kita-ku, Okayama, 700-8530, Japan 16 17 Running head 18 Amplitude, not period, has cline in circadian rhythm 19 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272070; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 20 Abstract 21 Many species show rhythmicity in activity, from the timing of flowering in 22 plants to that of foraging behaviour in animals. The free-running periods and 23 amplitude (sometimes called strength or power) of circadian rhythms are 24 often used as indicators of biological clocks. Many reports have shown that 25 these traits highly geographically variable, and interestingly, they often 26 show latitudinal or altitudinal clines. In many cases, the higher the latitude 27 is, the longer the free-running circadian period (i.e., period of rhythm) in 28 insects and plants. However, reports of positive correlations between latitude 29 or longitude and circadian rhythm traits, including free-running periods, the 30 power of the rhythm and locomotor activity, are limited to certain taxonomic 31 groups. Therefore, we collected a cosmopolitan stored-product pest species, 32 the red flour beetle Tribolium castaneum, in various parts of Japan and 33 examined its rhythm traits, including the power of the rhythm and period of 34 the rhythm, which were calculated from locomotor activity. The analysis 35 revealed that power was significantly lower for beetles collected in northern 36 areas compared with southern areas in Japan. However, it is worth noting 37 that the period of circadian rhythm did not show any clines; specifically, it 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272070; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 38 did not vary among the sampling sites, despite the very large sample size (n 39 = 1585). We discuss why these cline trends were observed in T. castaneum. 40 Keywords: biological clock, circadian rhythm, cline, latitude, longitude, 41 Tribolium castaneum, power of rhythm 42 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272070; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 43 Introduction 44 Latitudinal clines are of evolutionary interest because they indicate the 45 action of natural selection [1]. Many traits correlate with latitude, for 46 example, body size [2,3] and life history traits [4]. Circadian rhythm traits 47 are also correlated with latitude. 48 Circadian rhythms are particularly important for timing or regulating key 49 biological events in insects [5]. The free-running period and power of the 50 rhythm (i.e., sometimes called the amplitude or strength of the rhythm) are 51 often used as indicators of circadian rhythms [6,7], and there is much 52 evidence that the free-running periods of circadian rhythms exhibit 53 latitudinal or longitudinal clines at the phenotype to molecular levels in 54 many taxonomic groups [8-12]. 55 Additionally, in insect species other than Drosophila, many studies on the 56 relationships between free-running periods and latitude have been conducted. 57 In the linden bug, Pyrrhocoris apterus, higher-latitude populations are 58 reported to have longer free-running periods [13]. Similar positive relations 59 between latitude and circadian rhythms have been reported in a parasitic 60 wasp, Nasonia vitripennis [12], and also in plant species [14]. 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272070; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 61 In Drosophila species that evolved in tropical regions and then expanded 62 their distribution to temperate regions, rhythm traits vary depending on 63 where the flies live [15]. For example, pupal-adult eclosion rhythms in the 64 far north were more arrhythmic than those in the south among Drosophila 65 littoralis populations [16]. Circadian locomotor rhythms of D. melanogaster 66 derived from Africa had a stronger power of rhythm than those of more 67 northern Drosophila species in Europe [17]. However, pioneer studies 68 revealed a negative relation between latitude and these traits [18]. Overall, 69 these studies reveal that this topic remains controversial. 70 Given these discrepancies, we need more biological models. The red flour 71 beetle Tribolium castaneum (Herbst) [Tenebrionidae] is a cosmopolitan 72 stored-product pest [19], and hence, it can serve as an insect model species. 73 Its biology and behaviour are well studied [20,21]. Furthermore, genome 74 sequences are available for Tribolium castaneum [22]. 75 In Japan, this species is distributed in most areas except Hokkaido, in the 76 northern parts, meaning that it can be collected from a wide range of latitudes 77 [23]. It provides us with a novel, fascinating model with which to examine 78 the relations between latitude/longitude and circadian rhythm traits. Hence, 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272070; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 79 we studied the relationships between latitude or longitude and circadian 80 rhythm parameters, including the period of circadian rhythms, power of the 81 rhythm, and locomotor activity, in T. castaneum. 82 83 Materials and Methods 84 Insects 85 The geographical populations of Tribolium castaneum used in the present 86 study were collected from 38 different fields in Japan (Figure 1). Table S1 87 shows the latitude and longitude of the collection points, along with the 88 number of samples. The northernmost and southernmost points where the 89 insect could be collected were Aomori Prefecture (north latitude 40.89, east 90 longitude 127.69) and Kumamoto Prefecture-C (north latitude 32.57, east 91 longitude 130.66), respectively. The collection of insects was conducted 92 during 2016 and 2017. We collected beetles from each rice pearling mill in 93 Japan that was set up in a village or town with rice fields. More than twenty 94 beetles were collected from each mill. The collected beetles were reared with 95 a mixture of whole meal (Yoshikura Shokai, Tokyo) enriched with brewer’s 96 yeast (Asahi Beer, Tokyo) and maintained at 25°C with a 16 h photoperiod 6 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.28.272070; this version posted August 31, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 97 (lights on at 07:00, lights off at 23:00). Each collected group was kept in a 98 separate plastic Petri dish (90 mm in diameter, 15 mm in height). Since this 99 species is a stored-grain pest, these laboratory conditions were chosen to 100 closely mirror the native environment of these beetles. Before the 101 experiments, each beetle population was reared for more than two 102 generations in incubators (MIR-153, Sanyo, Osaka, Japan). We used beetles 103 reared for a few generations in the incubator in the experiment. 104 105 Locomotor activity 106 To assess circadian rhythm, we maintained beetles under 16L:8D conditions 107 for more than 20 days in an incubator kept at 25°C before the measurement 108 of locomotor activity, and we then measured the locomotor activity of T. 109 castaneum for 10 days in darkness. A beetle from each population was placed 110 in a clear plastic Petri dish (30 × 10 mm) in an incubator (MIR-153, Sanyo, 111 Osaka, Japan) maintained at 25°C under complete darkness (DD).