Circadian Rhythms of Locomotor Activity Mediated by Cryptochrome 2 and Period 1 Genes in the Termites Reticulitermes chinensis and Odontotermes formosanus

Simple Summary Almost all insects demonstrate a wide variety of behavioral, physiological, biochemical and molecular circadian rhythms during their natural selection process. Some examples of the circadian rhythm in insects include the sleep–wake cycle, foraging time, migration and hormone fluctuation. Although the circadian rhythms of activities have been studied in several termite species, the molecular mechanisms of circadian rhythms in termites are unclear. In this study, we observed that, even in complete darkness, the termites Reticulitermes chinensis and Odontotermes formosanus showed clear circadian rhythms of locomotor activity. Knockdown of the clock genes cryptochrome 2 (Cry2) and period 1 (Per1) impaired the circadian rhythms of locomotor activity in constant darkness in the two termite species. We verified that locomotor activity in subterranean termites is controlled by the circadian clock. Thus, this study contributes to a better understanding of circadian clock mechanisms in subterranean insects. Abstract Locomotor activity rhythms are crucial for foraging, mating and predator avoidance in insects. Although the circadian rhythms of activity have been studied in several termite species, the molecular mechanisms of circadian rhythms in termites are still unclear. In this study, we found that two termite species, R. chinensis and O. formosanus, exhibited clear circadian rhythms of locomotor activity in constant darkness along with rhythmically expressed core clock genes, Cry2 and Per1. The knockdown of Cry2 or Per1 expression in the two termite species disrupted the circadian rhythms of locomotor activity and markedly reduced locomotor activity in constant darkness, which demonstrates that Cry2 and Per1 can mediate the circadian rhythms of locomotor activity in termites in constant darkness. We suggest that locomotor activity in subterranean termites is controlled by the circadian clock.

In insects and mammals, it is well recognized that Cry, Per and Tim play a vital role in the endogenous circadian clock pathway [15][16][17].In the cockroach Blattella germanica, the knockdown of Cry2 severely disrupted the circadian rhythms of locomotor activity even in constant darkness (DD) [18].Under a 12 h light:12 h dark cycle (LD), mice exhibited clear circadian rhythms of locomotor activity, whereas with a double knockout of Cry1/Cry2 in mice they exhibited no clear circadian rhythms of locomotor activity [19].Similarly, mosquitoes show a rhythmicity of swarming to attract females for mating at certain times of the day [20], but knocking down the circadian clock genes Per and Tim disrupts male swarming behavior and limits their mating success [5].In the cricket Gryllus bimaculatus, the knockdown of Per impaired the circadian rhythm of locomotor activity under DD conditions [21].Thus, these examples indicate that the circadian clock genes Cry, Per and Tim can regulate the circadian rhythms of activities in insects and mammals.Notably, a recent study verified that Tim is lost in some social insects (nearly all termites) [22].In addition, light intensity, temperature and social interactions regulate the circadian rhythms of behavior and physiology in insects [23][24][25][26][27].Although the circadian rhythms of activities have been studied in several termite species (R. speratus, Macrotermes bellicosus, etc.) [28][29][30], the molecular mechanisms of circadian rhythm activity in subterranean termites are unclear.
The termites Reticulitermes chinensis and Odontotermes formosanus are widely distributed in China, especially in the area south of the Yangtze River [31].These two termite species make excellent candidates for studying circadian rhythms due to their irrelevant visual cues [32,33].In addition, the study of circadian rhythms in termites provides insight into the rhythmicity of subterranean insects and mammals.In order to investigate the behavioral and molecular mechanisms of activity rhythms in subterranean termites, we first tested the circadian rhythms of locomotor activity in the two termite species under different photoperiods.Second, we assessed the temporal expression patterns of clock genes in the heads of two termite species.Finally, we used RNA interference to analyze the influence of the clock genes Cry2 and Per1 on the circadian rhythms of locomotor activity in the two termite species.

Experimental Termites
We collected the termites R. chinensis and O. formosanus from Shizi Hill (Wuhan, China).The number of termite colonies used in this study is shown in Supplementary Tables S1 and S2.We transported these colonies of termites to the laboratory and then placed them in plastic boxes.Before the experiments, the termites were fed with pieces of moist filter paper for 3 days.Worker termites were used as experimental subjects in these experiments.The conditions for rearing the worker termites were as follows: 25 ± 1 • C and 70 ± 5% relative humidity (RH) in full darkness.

Measurement of Locomotor Activity
Termites were transferred to 9 cm Petri dishes (six worker termites per dish) with moist filter paper and then sealed with sealing film (it has been discovered in laboratories that moist filter paper can remain wet for more than 10 days).Before the experiments, termites were randomly divided into three different groups.One group was acclimated to constant darkness (DD) for 3 days, and the other two groups were acclimated to the light/dark cycle (LD, 12 L:12 D, illumination time 08:00-20:00 (Beijing Time, China); light intensity, 100 lux; irradiance, 0.62 W/m −2 ) or constant light (LL, light intensity, 100 lux; irradiance, 0.62 W/m −2 ) for 3 days, respectively (Figure 1A,B).During each experiment, we used a video camera with an infrared module (acA1920-40gc, Basler, Germany) to record Insects 2024, 15, 1 3 of 13 the circadian rhythms of the locomotor activity of worker termites.The camera captured video at 25 frames/s, and video analysis was conducted using the Noldus EthoVision tracking system (EthoVision XT 14, Wageningen, The Netherlands) [32][33][34].The distances moved by worker termites were measured in different photoperiods (DD, LD or LL) for 48 h (Figure 1A,B).The conditions of the trials were as follows: 25 ± 1 • C and 70 ± 5% RH in infrared illumination [35].
irradiance, 0.62 W/m -2 ) for 3 days, respectively (Figure 1A,B).During eac used a video camera with an infrared module (acA1920-40gc, Basler, Ge the circadian rhythms of the locomotor activity of worker termites.The video at 25 frames/s, and video analysis was conducted using the N tracking system (EthoVision XT 14, Wageningen, Netherlands) [32-3 moved by worker termites were measured in different photoperiods (D 48 h (Figure 1A,B).The conditions of the trials were as follows: 25 ± 1 °C in infrared illumination [35].

Expression Patterns of Clock Genes
Expression patterns of clock genes (Cry2 and Per1) were observed of the day (00:00, 04:00, 08:00, 12:00, 16:00 and 20:00) (Beijing Time, Chin LD conditions by qRT-PCR.We extracted the total RNA from the he termites using RNAiso Plus (Takara, Code No: 9109, Dalian, China).The cDNA templates using total RNA at the concentration of one microgram instructions of the PrimeScript TM RT Reagent Kit with gDNA Eraser (T RR047A, Dalian, China).Next, qRT-PCR was performed with the sy templates, gene-specific primers and SYBR Green Master Mix (High Rox Code No: 11203ES08, Shanghai, China) using the QuantStudio 6&7 Fle

Expression Patterns of Clock Genes
Expression patterns of clock genes (Cry2 and Per1) were observed at different times of the day (00:00, 04:00, 08:00, 12:00, 16:00 and 20:00) (Beijing Time, China) under DD and LD conditions by qRT-PCR.We extracted the total RNA from the heads of 10 worker termites using RNAiso Plus (Takara, Code No: 9109, Dalian, China).Then, we synthesized cDNA templates using total RNA at the concentration of one microgram according to the instructions of the PrimeScript TM RT Reagent Kit with gDNA Eraser (Takara, Code No: RR047A, Dalian, China).Next, qRT-PCR was performed with the synthesized cDNA templates, gene-specific primers and SYBR Green Master Mix (High Rox Plus) (YEASEN, Code No: 11203ES08, Shanghai, China) using the QuantStudio 6&7 Flex Real-Time PCR System (Applied Biosystems, Life Technologies Italia, MA, USA).Finally, mRNA levels were quantified using three genes (β-actin, Hsp 70 or NADH) as the reference genes [32,35].The primer sequences used for qRT-PCR are presented in Supplementary Table S3.The qRT-PCR data for the two clock genes Cry2 and Per1 were calculated via the 2 −∆∆CT method to analyze them [36].

Synthesis of dsRNA and Microinjection
Double-stranded Cry2 (dsCry2) and Per1 (dsPer1) were synthesized with the T7 transcription kit (Thermo Fisher Scientific, Code No: AM1354, MA, USA) according to the manufacturer's instructions.Firstly, PCR was carried out using the plasmids as templates in combination with specific primers (See Supplementary Table S4).Subsequently, with the purified PCR products as templates, T7 RNA Polymerase was used to generate dsRNA in transcription reactions.Next, dsRNA was dissolved in diethyl pyrocarbonate water and quantified by the NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, MA, USA).The dsRNA was subjected to 1% agarose gel, and the dsRNA solution was stored at −80 • C.
Two micrograms of dsRNA solution were injected into the side of the thorax in worker termites using a sterilized microinjector (World Precision Instruments, SYS-PV820, Florida, USA) [37,38].Termites were placed on moist filter paper within 9 cm Petri dishes after dsRNA injection.Ten worker termites were collected for measuring the mRNA of Cry2 or Per1 three days and five days after injection.In addition, three days after injection, the locomotor activities of worker termites were recorded for 48 h.An equivalent solution of double-stranded green fluorescent protein (dsGFP) was injected in control groups [39].

Statistical Analysis
The rhythms of locomotor activity over a 48 h period were determined using the cosinor procedure (http://www.circadian.org/softwar.html,accessed on 13 November 2023) [40].IBM SPSS Statistics 19.0 software was used to analyze the metric data.The normal distribution of the metric data was tested using the Shapiro-Wilk method.One-way analysis of variance (ANOVA) and Tukey's HSD test were used to analyze the temporal expression levels of genes and walking distances.The abnormal distribution of the metric data was tested using the Wilcoxon signed-rank test and Mann-Whitney test.The Wilcoxon rank-sum test was used to analyze the expression of the targeted genes Cry2 or Per1 after injection.Locomotor activity during the subjective day and subjective night was evaluated using the Mann-Whitney test.The significance level was set as p < 0.05.

Effects of Different Photoperiods on the Circadian Rhythms of Locomotor Activity in the Two Termite Species
We examined the effects of different photoperiods on the circadian rhythms of locomotor activity in the two termites R. chinensis and O. formosanus.We found that the termite R. chinensis exhibited a significant circadian rhythm of locomotor activity under DD conditions (p < 0.001) (Figure 2A).Similarly, the termite O. formosanus also exhibited a significant circadian rhythm of locomotor activity under DD conditions (p < 0.001) (Figure 3A).At the same time, the circadian periods of the two termites R. chinensis and O. formosanus were close to 24 h under DD conditions.However, under LD and LL conditions, the circadian rhythms of locomotor activity were abolished in the two termites R. chinensis (LD, p = 0.130; LL, p = 0.558) (Figure 2B,C) and O. formosanus (LD, p = 0.60; LL, p = 0.242) (Figure 3B,C).

Cry2 and Per1 Temporal Expression under DD Conditions in the Two Termite Species
We assayed head Cry2 and Per1 mRNA abundance over time in the two termit species by qRT-PCR in DD and LD conditions.Under DD conditions, we found that th expression of Cry2 and Per1 in the termite R. chinensis showed significant differences a different times of the day (ANOVA, Cry2, F(6,49) = 8.67, p < 0.001; Per1, F(6,49) = 4.42, p < 0.001 and the expression of Cry2 and Per1 in R. chinensis showed peak levels at 08:00-12:00 an its lowest level at 04:00 (Figure 4A,B).In the termite O. formosanus, we also found that th expression of Cry2 and Per1 showed significant differences at different times of the da (ANOVA, Cry2, F(6,49) = 11.18,p < 0.001; Per1, F(6,49) = 11.40,p < 0.001), and the expression o Cry2 and Per1 in O. formosanus showed peak levels at 12: 00 and its lowest level at 20:0 (Figure 4C,D).In addition, under LD conditions, we found that the expression of Cry2 an

Cry2 and Per1 Knockdown Disrupted the Circadian Rhythms of Locomotor A DD Conditions in the Two Termite Species
The injection of dsCry2 or dsPer1 caused a significant decrease in th mRNA level in the head of the termite R.   The injection of dsGFP (control) did not significantly affect the circadian locomotor activity under DD conditions in the termite R. chinensis (p < 0.001) However, the injection of dsCry2 or dsPer1 disrupted the circadian rhythms o activity (dsCry2, p = 0.152; dsPer1, p = 0.083) (Figure 6B,C).In addition, the dsCry2 significantly decreased locomotor activity compared with the co termite R. chinensis (ANOVA, F(2,32) = 4.34, p < 0.05) (Figure 6D).In the termite O the injection of dsGFP did not significantly impact the circadian rhythms o activity under DD conditions (p < 0.001) (Figure 6G), but the injection of dsCr disrupted the circadian rhythms of locomotor activity under DD conditions 0.128; dsPer1, p = 0.230) (Figure 6H,I).Moreover, the injection of dsCry significantly decreased locomotor activity compared with the control in th formosanus (ANOVA, F(2,51) = 21.68,p < 0.001) (Figure 6J).The injection of dsGFP (control) did not significantly affect the circadian rhythms of locomotor activity under DD conditions in the termite R. chinensis (p < 0.001) (Figure 6A).However, the injection of dsCry2 or dsPer1 disrupted the circadian rhythms of locomotor activity (dsCry2, p = 0.152; dsPer1, p = 0.083) (Figure 6B,C).In addition, the injection of dsCry2 significantly decreased locomotor activity compared with the control in the termite R. chinensis (ANOVA, F (2,32) = 4.34, p < 0.05) (Figure 6D).In the termite O. formosanus, the injection of dsGFP did not significantly impact the circadian rhythms of locomotor activity under DD conditions (p < 0.001) (Figure 6G), but the injection of dsCry2 or dsPer1 disrupted the circadian rhythms of locomotor activity under DD conditions (dsCry2, p = 0.128; dsPer1, p = 0.230) (Figure 6H,I).Moreover, the injection of dsCry2 or dsPer1 significantly decreased locomotor activity compared with the control in the termite O. formosanus (ANOVA, F (2,51) = 21.68,p < 0.001) (Figure 6J).

Discussion
Even though circadian rhythms are self-sustaining under constant conditions organisms (insects and mammals) nevertheless respond to environmental stimuli particularly illumination intensity and duration [41][42][43].In this study, our results showed that the two termites R. chinensis and O. formosanus exhibited no clear circadian rhythms of locomotor activity under LL and LD conditions, which is different to the pattern observed in other insects and mammals [18,44].In addition, since these two termite species have adapted to dark, underground environments, light may be a stressfu environment for subterranean termites, which indicates that light conditions impair the circadian rhythms of locomotor activity in termites.Thus, subterranean termites have

Discussion
Even though circadian rhythms are self-sustaining under constant conditions, organisms (insects and mammals) nevertheless respond to environmental stimuli, particularly illumination intensity and duration [41][42][43].In this study, our results showed that the two termites R. chinensis and O. formosanus exhibited no clear circadian rhythms of locomotor activity under LL and LD conditions, which is different to the pattern observed in other insects and mammals [18,44].In addition, since these two termite species have adapted to dark, underground environments, light may be a stressful environment for subterranean termites, which indicates that light conditions impair the circadian rhythms of locomotor activity in termites.Thus, subterranean termites have evolved different circadian rhythms of locomotor activity in response to their living environment.Surprisingly, under DD conditions, the termites R. chinensis and O. formosanus exhibited clear circadian rhythms of locomotor activity.Similarly, the cockroach Periplaneta americana exhibited a clear circadian rhythm of locomotor activity under complete darkness [18].The subterranean vole Lasiopodomys mandarinus (eusocial mammal) also exhibited a clear circadian rhythm of locomotor activity under DD conditions [44].These observations reflect the adaptation of subterranean termites, cockroaches, and voles to dark underground environments.Because the circadian rhythms of locomotor activity are exhibited even in constant conditions, we suspect that the endogenous circadian clock is important for the regulation of the circadian rhythms of locomotor activity in termites.
In insects and mammals, the expression of clock genes fluctuated rhythmically in a 24 h period [24,45].In this study, the Cry2 levels of two termite species exhibited a clear circadian rhythm, with higher levels during subjective day and lower levels during subjective night under DD conditions, which exactly coincided with the circadian rhythm of locomotor activity in two termite species.Per1 levels showed a similar trend to Cry2, exhibiting a clear circadian rhythm.The Cry and Per genes are established clock core complex molecules [46,47].We conclude that Cry2 and Per1 genes may play a critical role in the circadian rhythm of locomotor activity in these two termite species.
Knockdown of Cry2 or Per1 significantly impaired the circadian rhythms of locomotor activity and markedly reduced locomotor activity under DD conditions in R. chinensis and O. formosanus.Analogously, knockdown of Cry2 severely disrupted the circadian rhythms of locomotor activity under DD conditions in the cockroach B. germanica [18].Silencing of Per in the cricket G. bimaculatus completely disrupted the circadian rhythm of locomotor activity in constant darkness [21].In mammals, Cry2 mutation caused a shortened circadian period and impaired behavioral rhythms under a light-dark cycle in mice [19].We suggest that the two core clock genes Cry2 and Per1 can regulate the circadian rhythms of locomotor activity under DD conditions in termites.Furthermore, knockdown of Cry2 or Per1 in R. chinensis and O. formosanus markedly reduced locomotor activity during the day, which indicates that Cry2 and Per1 are essential for the maintenance of termite circadian rhythms and their locomotor behavior.

Conclusions
Taken together, our study delivers evidence that the two termites R. chinensis and O. formosanus exhibit clear circadian rhythms of locomotor activity under DD conditions.However, under LD and LL conditions, complete loss of circadian rhythms of locomotor activity was observed in these two termites.Subterranean termites' foraging activities mainly occur during the subjective day.Strikingly, the clock genes Cry2 and Per1 display cyclical expression in the heads of termites under DD conditions.In addition, knockdown of Cry2 and Per1 disrupted the circadian rhythms of locomotor activity in the two termites, which suggests that Cry2 and Per1 participate in termite circadian rhythms under DD conditions.Thus, this study contributes to a better understanding of circadian clock mechanisms in subterranean insects.

Supplementary Materials:
The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/insects15010001/s1, Figure S1: The patterns of Cry2 and Per1 gene expression in the two termite species; Table S1: The distribution of the four colonies of R. chinensis for each experiment; Table S2: The distribution of the seven colonies of O. formosanus for each experiment; Table S3: Primers used for qRT-PCR analyses; Table S4

Figure 1 .
Figure 1.Experimental settings and tracking method for the termites.(A) Experi quantitative system for analyzing behavior of termites.The video recordings mpeg format for analysis of the parameters using the EthoVision video tr Schematic diagram of the timeline for the behavioral assay.

Figure 1 .
Figure 1.Experimental settings and tracking method for the termites.(A) Experimental setup of the quantitative system for analyzing behavior of termites.The video recordings were converted to mpeg format for analysis of the parameters using the EthoVision video tracking system.(B) Schematic diagram of the timeline for the behavioral assay.

Figure 2 .
Figure 2. The circadian rhythms of locomotor activity in the termite R. chinensis under differen photoperiods.(A-C) Locomotor activity of the termite R. chinensis under DD (n = 10) (A), LD (n = 9 (B) and LL (n = 9) (C) photoperiod conditions.The data in the figures are mean ± SEM, and the circadian rhythms of locomotor activity were analyzed using the cosinor procedure (A-C).(D) The total walking distances of the termite R. chinensis during a 48 h experimental period.The data in the figures are mean ± SEM, and different letters express significant differences according to Tukey' HSD test (p < 0.05) (D).(E-G) The total walking distances of the termite R. chinensis in subjective day and subjective night under DD (E), LD (F) and LL (G) photoperiod conditions.The data represent mean ± SEM.Asterisks indicate significant differences determined by the Mann-Whitney test (** p < 0.01; *** p < 0.001) (E-G).The horizontal bars represent the light conditions during the experiment.White boxes represent light and black and gray boxes represent dark in (A-C).

Figure 2 .
Figure 2. The circadian rhythms of locomotor activity in the termite R. chinensis under different photoperiods.(A-C) Locomotor activity of the termite R. chinensis under DD (n = 10) (A), LD (n = 9) (B) and LL (n = 9) (C) photoperiod conditions.The data in the figures are mean ± SEM, and the circadian rhythms of locomotor activity were analyzed using the cosinor procedure (A-C).(D) The total walking distances of the termite R. chinensis during a 48 h experimental period.The data in the figures are mean ± SEM, and different letters express significant differences according to Tukey's HSD test (p < 0.05) (D).(E-G) The total walking distances of the termite R. chinensis in subjective day and subjective night under DD (E), LD (F) and LL (G) photoperiod conditions.The data represent mean ± SEM.Asterisks indicate significant differences determined by the Mann-Whitney test (** p < 0.01; *** p < 0.001) (E-G).The horizontal bars represent the light conditions during the experiment.White boxes represent light and black and gray boxes represent dark in (A-C).

Figure 3 .
Figure 3.The circadian rhythms of locomotor activity in the termite O. formosanus under differen photoperiods.(A-C) Locomotor activity of the termite O. formosanus under DD (n = 10) (A), LD (n 9) (B) and LL (n = 9) (C) photoperiod conditions.The data in the figures are mean ± SEM, and th circadian rhythms of locomotor activity were analyzed using the cosinor procedure (A-C).(D) Th total walking distances of the termite O. formosanus during a 48 h experimental period.The data i the figures are mean ± SEM, and different letters express significant differences according to Tukey HSD test (p < 0.05) (D).(E-G) The total walking distances of the termite O. formosanus in subjectiv day and subjective night under DD (E), LD (F) and LL (G) photoperiod conditions.The dat represent mean ± SEM.Asterisks indicate significant differences determined by the Mann-Whitne test (*** p < 0.001) (E-G).

Figure 3 .
Figure 3.The circadian rhythms of locomotor activity in the termite O. formosanus under different photoperiods.(A-C) Locomotor activity of the termite O. formosanus under DD (n = 10) (A), LD (n = 9) (B) and LL (n = 9) (C) photoperiod conditions.The data in the figures are mean ± SEM, and the circadian rhythms of locomotor activity were analyzed using the cosinor procedure (A-C).(D) The total walking distances of the termite O. formosanus during a 48 h experimental period.The data in the figures are mean ± SEM, and different letters express significant differences according to Tukey's HSD test (p < 0.05) (D).(E-G) The total walking distances of the termite O. formosanus in subjective day and subjective night under DD (E), LD (F) and LL (G) photoperiod conditions.The data represent mean ± SEM.Asterisks indicate significant differences determined by the Mann-Whitney test (*** p < 0.001) (E-G).

Figure 4 .
Figure 4.The patterns of Cry2 and Per1 gene expression in two termite species.(A) Cry2 expression in the termite R. chinensis under DD conditions (n = 8).(B) The expression in the termite R. chinensis under DD conditions (n = 8).(C) The p expression in the termite O. formosanus under DD conditions (n = 8).(D) The expression in the termite O. formosanus under DD conditions (n = 8).The data in th mean ± SEM, and different letters express significant differences according to Tuke 0.05).The mRNA levels were normalized relative to the two termite species collecte and black bars represent subjective day and subjective night, respectively.

Figure 4 .
Figure 4.The patterns of Cry2 and Per1 gene expression in two termite species.(A) The patterns of Cry2 expression in the termite R. chinensis under DD conditions (n = 8).(B) The patterns of Per1 expression in the termite R. chinensis under DD conditions (n = 8).(C) The patterns of Cry2 expression in the termite O. formosanus under DD conditions (n = 8).(D) The patterns of Per1 expression in the termite O. formosanus under DD conditions (n = 8).The data in the figures are the mean ± SEM, and different letters express significant differences according to Tukey's HSD test (p < 0.05).The mRNA levels were normalized relative to the two termite species collected at 04:00.Gray and black bars represent subjective day and subjective night, respectively.

Insects 2023 , 12 Figure 6 .
Figure 6.Knockdown of Cry2 and Per1 disrupted the circadian rhythms of locomotor activities in the two termite species.(A-C) The circadian rhythms of locomotor activities of the termite R chinensis injected with dsGFP (n = 12) (A), dsCry2 (n = 12) (B), or dsPer1 (n = 12) (C) were assessed 3 days after injection.(D) The walking distances of the termite R. chinensis after 3 days of dsRNA injections during a 48 h experimental period.(E,F) The walking distances of the termite R. chinensi 3 days after injecting dsRNA during subjective day (E) and night (F).(G-I) The circadian rhythm of locomotor activities of the termite O. formosanus injected with dsGFP (n = 18) (G), dsCry2 (n = 18 (H), or dsPer1 (n = 18) (I) were assessed 3 days after injection.(J) The walking distances of the termite O. formosanus after 3 days of dsRNA injections during a 48 h experimental period.(K,L) The walking distances of the termite O. formosanus 3 days after injecting dsRNA during subjective day (K) and night (L).The rhythmicity of locomotor activity was analyzed using the cosinor procedure (A-C,G-I).The data in the figures are mean ± SEM, and different letters express significant difference according to Tukey's HSD test (p < 0.05) (D-F,J-L).Gray and black bars represent subjective day and subjective night, respectively (A-C,G-I).

Figure 6 .
Figure 6.Knockdown of Cry2 and Per1 disrupted the circadian rhythms of locomotor activities in the two termite species.(A-C) The circadian rhythms of locomotor activities of the termite R. chinensis injected with dsGFP (n = 12) (A), dsCry2 (n = 12) (B), or dsPer1 (n = 12) (C) were assessed 3 days after injection.(D) The walking distances of the termite R. chinensis after 3 days of dsRNA injections during a 48 h experimental period.(E,F) The walking distances of the termite R. chinensis 3 days after injecting dsRNA during subjective day (E) and night (F).(G-I) The circadian rhythms of locomotor activities of the termite O. formosanus injected with dsGFP (n = 18) (G), dsCry2 (n = 18) (H), or dsPer1 (n = 18) (I) were assessed 3 days after injection.(J) The walking distances of the termite O. formosanus after 3 days of dsRNA injections during a 48 h experimental period.(K,L) The walking distances of the termite O. formosanus 3 days after injecting dsRNA during subjective day (K) and night (L).The rhythmicity of locomotor activity was analyzed using the cosinor procedure (A-C,G-I).The data in the figures are mean ± SEM, and different letters express significant differences according to Tukey's HSD test (p < 0.05) (D-F,J-L).Gray and black bars represent subjective day and subjective night, respectively (A-C,G-I).