Sensory conflict disrupts circadian rhythms in the sea anemone Nematostella vectensis
- Biology Department, Woods Hole Oceanographic Institution, United States
- MIT-WHOI Joint Program in Oceanography/Applied Ocean Science & Engineering, United States
Figures
Figure 1
Schematic of the experimental design.
(A) Anemones in constant darkness were entrained to ramped 24 hr temperature cycles that changed gradually from 14–26 °C, or from 8–32 °C. For temperature cycle experiments, ZT0 refers to the …
Figure 2 with 1 supplement
Temperature cycles drive rhythmic locomotor behavior and Cry2 expression, and weakly entrain circadian behavior.
(A) Mean behavior profiles of anemones in each group over 3 days. Individual locomotor profiles were normalized, averaged, and smoothed (see Materials and methods). Shaded area represents standard …
-
Figure 2—source data 1
Behavioral rhythmicity results.
- https://cdn.elifesciences.org/articles/81084/elife-81084-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Zeitgeber cycles with a 12 hr period entrain circadian rhythms in Nematostella.
(A) Mean behavior profile (left), average 24 hr behavior profile (middle), and Lomb–Scargle periodogram (right) of animals entrained in a 6:6 LD cycle (n=12). (B) Mean behavior profile (left), …
Figure 3 with 2 supplements
Sensory conflict disrupts rhythmic locomotor behavior in Nematostella.
(A) Mean behavior profiles. Individual locomotor profiles were normalized, averaged, and smoothed (see Materials and methods). Shaded area represents standard error. Left, entrainment conditions; …
Figure 3—figure supplement 1
Period lengths of rhythmic individuals.
Periods had a wider variance in free-running groups compared to entrainment conditions (Levene’s test for variance homogeneity, p=1 × 10−4). However, there was no significant difference in mean …
Figure 3—figure supplement 2
Lomb-Scargle Periodogram (LSP) power across groups.
*: Significantly lower than Aligned group (Dunn test, p<0.05).
Figure 4
Clustering analysis of mean behavior profiles based on wavelet transformation.
(A) Principal component analysis of wavelet transformation distance matrix. Time series were grouped and colored based on the clusters identified in (B). C: cycles; FR: free-running. (B) …
Figure 5 with 1 supplement
Sensory conflict alters patterns of rhythmic gene expression.
(A) Discriminant analysis of principal components (DAPC) demonstrates shifts in diel gene expression under SC. Density plot of sample loading values of light and dark samples during Aligned and SC …
-
Figure 5—source data 1
Counts matrix.
Contains TMM-normalized counts-per-million on a log2 scale.
- https://cdn.elifesciences.org/articles/81084/elife-81084-fig5-data1-v2.txt
-
Figure 5—source data 2
Differential expression, rhythmicity, and WGCNA analyses, and gene annotations.
- https://cdn.elifesciences.org/articles/81084/elife-81084-fig5-data2-v2.xlsx
-
Figure 5—source data 3
Results of HOMER motif enrichment analyses.
- https://cdn.elifesciences.org/articles/81084/elife-81084-fig5-data3-v2.xlsx
-
Figure 5—source data 4
GO and KEGG enrichment results.
- https://cdn.elifesciences.org/articles/81084/elife-81084-fig5-data4-v2.xlsx
Figure 5—figure supplement 1
Amplitude and mean expression of rhythmic genes.
(A) Relative amplitude (midpoint to peak) of shared rhythmic genes was reduced during SC. (B) Mean expression level of condition-specific rhythmic genes was lower during SC. Significance calculated …
Figure 6 with 1 supplement
Network analysis identifies changes to rhythmically co-expressed gene modules.
(A) Expression patterns of select modules in the full network. Black lines are Z-scores (difference from mean divided by standard deviation) of the expression of each gene, and the colored line is …
Figure 6—figure supplement 1
WGCNA module expression, Full network.
Black lines are Z-scores of the expression of each gene, and colored lines are means of those Z-scores. Left panels show module expression in Aligned samples, and right panels show expression in SC …
Videos
Video 1
Example of Nematostella vectensis locomotor behavior, with tracked center-point estimated by DeepLabCut.
This video is sped up 200 x and covers a period of 100 min. Width of arena is 3.5 cm.
Tables
Table 1
Summary statistics for sensory conflict experiments: Entrainment groups.
| Group | LSP p-value* | LSP power | eJTK p-value† | n rhythmic‡ | Period § | Phase § | Mean phase ¶ | Phase variance** | Amplitude†† |
|---|---|---|---|---|---|---|---|---|---|
| Aligned-cycle | 7.9e-4 | 0.85 | 2e-5 | 20/24 | 23.60 | 19.29 | 18.04 | 0.34 | 0.091 |
| Off2-cycle | 7.9e-4 | 0.75 | 2e-5 | 20/24 | 23.48 | 18.71 | 18.42 | 0.24 | 0.12 |
| Off4-cycle | 7.9e-4 | 0.84 | 2e-5 | 18/24 | 23.76 | 18.90 | 17.70 | 0.27 | 0.12 |
| Off6-cycle | 7.9e-4 | 0.60 | 2e-5 | 16/24 | 24.30 | 20.16 | 21.68 | 0.15 | 0.075 |
| Off8-cycle | 7.9e-4 | 0.85 | 2e-5 | 15/24 | 23.60 | 19.20 | 19.12 | 0.26 | 0.085 |
| Off10-cycle | 3.6e-3 | 0.19 | 1.7e-4 | 10/24 | 22.60 | 17.75 | 15.15 | 0.45 | 0.042 ‡ ‡ |
| Off12-cycle | 7.9e-4 | 0.22 | 4.5e-3 | 13/36 | 23.66 | 18.62 | 13.29 | 0.86 | 0.015 ‡ ‡ |
-
*
Tested periods between 20–28 hr.
-
†
Tested period of 24 hr.
-
‡
eJTK p<1 × 10−3.
-
§
determined by MFourFit.
-
¶
Circular mean of the phases of rhythmic individuals (eJTK p<1 × 10−3).
-
**
Circular variance of the phases of rhythmic individuals (eJTK p<1 × 10−3).
-
††
Determined with CircaCompare.
-
‡ ‡
:Reduced amplitude compared to Aligned-cycle, CircaCompare, p<0.05. LSP: Lomb-Scargle periodogram.
Table 2
Summary statistics for sensory conflict experiments: Free-running groups.
| Group | LSP p-value* | LSP power | eJTK p-value† | n rhythmic‡ | Period § | Phase § | Mean phase ¶ | Phase variance** | Amplitude†† |
|---|---|---|---|---|---|---|---|---|---|
| Aligned-FR | 7.9e-4 | 0.47 | 7e-5 | 10/24 | 25.84 | 11.63 | 12.67 | 0.35 | 0.060 |
| Off2-FR | 7.9e-4 | 0.79 | 7e-5 | 6/24 | 22.62 | 16.50 | 18.28 | 0.63 | 0.071 |
| Off4-FR | 7.9e-4 | 0.51 | 7e-5 | 11/24 | 23.64 | 14.17 | 6.98 | 0.74 | 0.050 |
| Off6-FR | 0.13 | 0.095 | 0.76 | 5/24 | 27.34 | 18.57 | 3.50 | 0.73 | 4.9e-3 ‡ ‡ |
| Off8-FR | 7.9e-4 | 0.67 | 7e-5 | 11/24 | 21.74 | 15.53 | 13.65 | 0.51 | 0.048 |
| Off10-FR | 0.53 | 0.042 | 0.61 | 7/24 | 28.00 | 3.39 | 10.78 | 0.70 | 2.8e-3 ‡ ‡ |
| Off12-FR | 7.9e-4 | 0.32 | 7e-5 | 6/24 | 22.96 | 11.72 | 20.65 | 0.80 | 0.034 ‡ ‡ |
-
*
Tested periods between 20–28 hr.
-
†
Tested period of 24 hr.
-
‡
eJTK p<1 × 10−3.
-
§
determined by MFourFit.
-
¶
Circular mean of the phases of rhythmic individuals (eJTK p<1 × 10−3).
-
**
Circular variance of the phases of rhythmic individuals (eJTK p<1 × 10−3).
-
††
Determined with CircaCompare.
-
‡ ‡
Reduced amplitude compared to Aligned-FR, CircaCompare, p<0.05. LSP: Lomb-Scargle periodogram.
Table 3
Comparison of phases of putative clock genes.
| Study | Clock | Helt | CIPC | PAR-bZIP-a | PAR-bZIP-c | Cry1a | Cry1b | Cry2 |
|---|---|---|---|---|---|---|---|---|
| Current study (Aligned) | 12.4 | 9.2 | 1.0 | 7.3 | 22.3 | 9.3 | 10.5 | 5.6 |
| Current study (SC) | 11.94 | 7.9 | 19.3 | 8.0 | 16.2 | 12.1 | NA | 21.8 |
| Oren et al., 2015* | 10.9 | 7.3 | 23.4 | 5.3 | 19.4 | 7.9 | NA | 2.3 |
| SimrBase ID | 6258 | 15017 | 4651 | 8136 | 8448 | 8109 | 8041 | 15282 |
| JGI ID | 160110 | 246249 | 245026 | 150375/ 87565 | 39846 | 168581 | 106062 | 194898 |
-
Phases estimated with CircaCompare. NA: below significance cutoff, phase not calculated. We used a p-value cutoff of p=0.01 for the current study, and p=0.05 for (Oren et al., 2015) because of their small sample size.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Commercial assay or kit | Aurum Total RNA Fatty and Fibrous Tissue Kit | Bio-Rad | #7326830 | |
| Commercial assay or kit | iScript cDNA synthesis kit | Bio-Rad | #1708891 | |
| Commercial assay or kit | iTaq Universal SYBR Green Supermix | Bio-Rad | #1725120 | |
| Commercial assay or kit | Aurum Total RNA Mini Kit | Bio-Rad | #736820 | |
| Software, algorithm | LinRegPCR | Ruijter et al., 2009 | ||
| Software, algorithm | NORMA-Gene | Heckmann et al., 2011 | ||
| Software, algorithm | DeepLabCut | Mathis et al., 2018 | ||
| Software, algorithm | R | R Project for Statistical Computing | RRID:SCR_001905 | |
| Sequence-based reagent | Clock_F | This paper | PCR primers | TAACCCGGAAGCTGA ATTTG |
| Sequence-based reagent | Clock_R | This paper | PCR primers | GCTTGGGGAAGACAC TAACTTG |
| Sequence-based reagent | Cry2_F | This paper | PCR primers | GCATCTGATTTGCA GAAATGG |
| Sequence-based reagent | Cry2_R | This paper | PCR primers | CTACACGGGCGA GATAGTGG |
| Sequence-based reagent | Cry1a_F | This paper | PCR primers | GCATGAATTCTG GCAGCTGG |
| Sequence-based reagent | Cry1a_R | This paper | PCR primers | CCAACTTCCACA GGGCAGAA |
| Sequence-based reagent | Cry1b_F | This paper | PCR primers | GATTCGGATGTTT GTCGCCA |
| Sequence-based reagent | Cry1b_R | This paper | PCR primers | TCGAACGAGTCCAG TGAACA |
| Sequence-based reagent | Helt_F | This paper | PCR primers | CGGACAAGGGCGCT AATGAA |
| Sequence-based reagent | Helt _R | This paper | PCR primers | CAAGGCTGTTGAGGG TCCAT |
