Interdependence between SEB-3 receptor and NLP-49 peptides shifts across predator-induced defensive behavioral modes in Caenorhabditis elegans
Figures

C. elegans responses to predatory threat can be organized into three defense modes.
(A) Predatory imminence model of C. elegans defensive responses to a predator-inhabited bacterial food patch. Upon being bitten by a predator, C. elegans executes an escape response, exits the predator-inhabited patch, and then ultimately reenters the patch (circa-strike mode). After extended exposure, C. elegans adopts an outstretched feeding posture to minimize predator contact (post-encounter mode). When confronted with a new patch, the predator-exposed C. elegans cautiously explores for potential predators in the patch (pre-encounter mode). (B) Arena setup for assessing circa-strike and post-encounter behaviors includes a 9.5 mm circular arena with a 2 mm bacterial patch, housing one C. elegans and four RS5194 P. pacificus predators (or none). (C) Number of exits and (D) total time that C. elegans spent off the patch during 1 hour exposure to predator and predator-free conditions (Dunn’s test, nC. elegans = 13). (E) Percentage of C. elegans animals adopting outstretched feeding posture across different exposure durations to both predator and predator-free conditions (Fisher’s exact test, nC. elegans = 44–45). (F) Arena setup for studying pre-encounter behaviors involved placing one C. elegans and four RS5194 P. pacificus predators (or none) in either a wide exit arena (open space around the food patch) or a narrow exit arena (narrow corridor to/from the food patch) with a 2 mm bacterial patch. After 2- or 4-hour predator exposure, C. elegans was transferred to a predator-free arena for a 15-minute exploration period. (G) Latency to enter a new patch (Dunn’s test with Benjamini–Hochberg adjustment, nC. elegans = 21–24) and (H) number of bins explored by C. elegans following either 2- or 4-hour exposure to predator or predator-free conditions (Welch’s t-test with Benjamini–Hochberg adjustment, nC. elegans = 13–15). Error bars are 95% bootstrap CIs containing the mean. n.s. = p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
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Figure 1—source data 1
Data for Figure 1C and D.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig1-data1-v1.zip
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Figure 1—source data 2
Data for Figure 1E and Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig1-data2-v1.zip
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Figure 1—source data 3
Data for Figure 1G.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig1-data3-v1.zip
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Figure 1—source data 4
Data for Figure 1H.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig1-data4-v1.zip

Images of arena setups.
(A) Wide exit arena. A 9.5 mm wide arena contains a 2 mm wide bacterial food patch as well as one adult C. elegans (arrow) and four adult Pristionchus sp. predators. (B) Representative example of C. elegans adopting outstretched feeding posture (arrow), with only its head in contact with bacteria. The typical sinusoidal waveform observed in (A) is distorted in the outstretched feeding posture. (C) Narrow exit arena. A dumbbell-shaped arena consisting of a pair of 2 mm wide circular cutouts separated by a 3 mm × 0.7 mm corridor. One circular cutout is completely filled with a 2 mm wide bacterial food patch. (D). Example image of a 10 × 10 square grid overlaid on top of the patch for counting bins explored by C. elegans.

Progression of post-encounter behavior acquisition.
Percentage of C. elegans animals adopting outstretched feeding posture at different hours of exposure to RS5194 P. pacificus (Fisher’s exact test with Benjamini–Hochberg adjustment, nC. elegans = 45).

Pre-encounter behavior is reversible and not explained by injuryinduced changes to locomotor speed.
(A–C) The pre-patch period is defined as starting from the initial placement of C. elegans in the empty circular cutout of a narrow exit arena and lasting until its mouth touches the new patch. (A) Distance traveled in the pre-patch period and (B) duration of the pre-patch period for C. elegans exposure either to RS5194 P. pacificus predators or no predators (Dunn’s test, nC. elegans = 38–41). (C) Association between pre-patch distance and pre-patch duration. Bold lines represent linear regression lines, with shaded regions representing 95% CIs from linear regression models. Pre-patch speed was estimated as the slopes of regression lines. (Kruskal–Wallis test on residuals of linear regression models, nC. elegans = 38–41). (D) Bins explored immediately or 6 hours after 4-hour exposure to either RS5194 P. pacificus predators or no predators (Student’s t-test and paired t-test with Benjamini–Hochberg adjustment, nC. elegans = 8–9). Error bars are 95% bootstrap CIs containing the mean. n.s. = p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
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Figure 1—figure supplement 3—source data 1
Data for Figure 1—figure supplement 3A–C.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig1-figsupp3-data1-v1.zip
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Figure 1—figure supplement 3—source data 2
Data for Figure 1—figure supplement 3D.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig1-figsupp3-data2-v1.zip

Post-encounter and pre-encounter behaviors are not explained by food deprivation.
(A) Percentage of C. elegans animals adopting outstretched feeding posture at different hours of exposure to RS5194 P. pacificus or food deprivation (Fisher’s exact test, nC. elegans = 26–31). (B) Latency to enter a new patch following 4-hour exposure to RS5194 predators, food deprivation, or neither (Dunn’s test with Benjamini–Hochberg adjustment, nC. elegans = 23–31). (C) Number of bins explored by C. elegans following 4-hour exposure to RS5194 predators, food deprivation, or neither (Dunn’s test with Benjamini–Hochberg adjustment, nC. elegans = 7–20). Error bars are 95% bootstrap CIs containing the mean. n.s. = p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. @par.
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Figure 1—figure supplement 4—source data 1
Data for Figure 1—figure supplement 4A.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig1-figsupp4-data1-v1.zip
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Figure 1—figure supplement 4—source data 2
Data for Figure 1—figure supplement 4B.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig1-figsupp4-data2-v1.zip
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Figure 1—figure supplement 4—source data 3
Data for Figure 1—figure supplement 4C.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig1-figsupp4-data3-v1.zip

Defensive response intensity increases with predation risk.
(A) Schematic of predatory harm potential of various Pristionchus spp. predator strains, based on studies previously conducted by this lab. (B) Number of bite-induced escape responses during 1-hour exposure to Pristionchus spp. predators (Dunn’s test with Benjamini–Hochberg adjustment, nC. elegans = 13–25). (C) Probability of exit following an escape response (binomial logistic regression followed by Wald test with single-step adjustment for Tukey contrasts, nC. elegans = 10–25). (D) Latency to reenter the patch following an exit, averaged across escape-induced exits, for various predator strains (Kruskal–Wallis test, nC. elegans = 10–25). (E) Percentage of C. elegans animals adopting outstretched feeding posture across different exposure durations to various predator conditions (Fisher’s exact test with Benjamini–Hochberg adjustment, nC. elegans = 36–45). Pairwise comparisons between predator strains are displayed on the right. (F) Latency to enter a new patch (Dunn’s test with Benjamini–Hochberg adjustment, nc. elegans = 22–40) and (G) bins explored (Dunn’s test with Benjamini-Hochberg adjustment, nC. elegans = 11–28) following 4-hour exposure to various predator conditions. Error bars in (C) are predicted P(exit | escape) and 95% CIs from binomial logistic regression model of data. All other error bars are 95% bootstrap CIs containing the mean. n.s. = p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
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Figure 2—source data 1
Data for Figure 2B–D, Figure 2—figure supplement 1B and C.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig2-data1-v1.zip
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Figure 2—source data 2
Data for Figure 2E.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig2-data2-v1.zip
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Figure 2—source data 3
Data for Figure 2F.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig2-data3-v1.zip
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Figure 2—source data 4
Data for Figure 2G, Figure 2—figure supplement 2A and B.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig2-data4-v1.zip

Patch exit latency is unaffected by predator strain.
(A) Total number of exits, with or without preceding bite, in various predator and predator-free conditions (Dunn’s test with Benjamini–Hochberg adjustment, nC. elegans = 13–25). (B) Latency between bite-induced escape response and subsequently exiting the patch, averaged across escape-induced exits, for various predator strains (Kruskal–Wallis test, nC. elegans = 10–25). (C) Probability of an aborted escape given that the escape did not result in an exit (binomial logistic regression followed by Wald test with single-step adjustment for Tukey contrasts, nC. elegans = 9–18). Error bars are 95% bootstrap CIs containing the mean. n.s. = p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
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Figure 2—figure supplement 1—source data 1
Data for Figure 2—figure supplement 1A.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig2-figsupp1-data1-v1.zip

Effect of extended exposure to various predations on pre-encounter behavior.
(A, B) Number of bins explored by C. elegans following (A) 6-hour (Tukey’s test, nC. elegans = 15–21) and (B) 8-hour (Dunn’s test with Benjamini–Hochberg adjustment, C. elegans = 15–20) exposure to various predator conditions. Error bars are 95% bootstrap CIs containing the mean. n.s. = p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

SEB-3 and NLP-49 peptides differentially regulate defense modes.
(A, B) Probability of exit following an escape response by (A) seb-3 strains (nC. elegans = 17–34) and (B) nlp-49 strains (nC. elegans = 15–18) (binomial logistic regression followed by Wald test with single-step adjustment for Tukey contrasts). (C, D) Latency to reenter the patch following an exit, averaged across escape-induced exits, for (C) seb-3 strains (nC. elegans = 17–34) and (D) nlp-49 strains (nC. elegans = 14–18) (Dunn’s test with Benjamini–Hochberg adjustment). (E, F) Percentage of animals adopting outstretched feeding posture in (E) seb-3 strains (nC. elegans = 27–63) and (F) nlp-49 strains (nC. elegans = 31–33) (Fisher’s exact test with Benjamini–Hochberg adjustment). (G, H) Bins explored following 4-hour exposure to predator or predator-free conditions, by (G) seb-3 strains (Wilcoxon’s ranked sum test with Benjamini–Hochberg adjustment, nC. elegans = 16–18) and (H) nlp-49 strains (Welch’s t-test with Benjamini–Hochberg adjustment, nC. elegans = 9–12). Error bars in (A, B) are predicted P(exit | escape) and 95% CIs from binomial logistic regression model of data. All other error bars are 95% bootstrap CIs containing the mean. n.s. = p > 0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
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Figure 3—source data 1
Data for Figure 3A and C, Figure 3—figure supplements 1B and 2A, C.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig3-data1-v1.zip
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Figure 3—source data 2
Data for Figure 3B and D, Figure 3—figure supplements 1C and 2B, D.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig3-data2-v1.zip
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Figure 3—source data 3
Data for Figure 3E.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig3-data3-v1.zip
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Figure 3—source data 4
Data for Figure 3F.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig3-data4-v1.zip
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Figure 3—source data 5
Data for Figure 3H.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig3-data5-v1.zip
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Figure 3—source data 6
Data for Figure 3H.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig3-data6-v1.zip

Baseline speeds of seb-3 and nlp-49 strains.
(A) Image of arena setup for assaying baseline on-bacteria escape speed. A 2-mm-wide arena is completely filled with a 2-mm-wide bacterial food patch. One adult C. elegans (arrow) and four adult RS5194 P. pacificus were placed in the arena and recorded for 2 hours. (B, C) Histogram of pooled mean latencies between escape response and exit from the patch for (B) seb-3 strains and their controls (nC. elegans = 80) and (C) nlp-49 strains and their controls (nC. elegans = 49). Based on theses histograms, escape speed was evaluated within the 15-second period following a bite. (D, E) Bite escape speed across consecutive bites in (D) seb-3 strains (nC. elegans = 20-27) and (E) nlp-49 strains (nC. elegans = 11-13). Bold lines indicate mean speed over a 5-bite sliding window, with shaded areas denoting bootstrap 95% CIs. Significance (*p<0.05) determined by non-overlapping CIs between wildtype and other strains. (F, G) Baseline speed in bacteria-free, predator-free conditions for (F) seb-3 strains (Tukey’s test, nC. elegans = 10) and (G) nlp-49 strains (one-way ANOVA, nC. elegans = 9–10). Error bars are 95% bootstrap CIs containing the mean. n.s. = p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
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Figure 3—figure supplement 1—source data 1
Data for Figure 3—figure supplement 1D.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
Data for Figure 3—figure supplement 1E.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig3-figsupp1-data2-v1.zip
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Figure 3—figure supplement 1—source data 3
Data for Figure 3—figure supplement 1F, G, Figure 4—figure supplement 2A.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig3-figsupp1-data3-v1.zip

Escapes and exit latencies by seb-3 and nlp-49 strains.
(A, B) Number of bite-induced escape responses executed by (A) seb-3 strains (Kruskal–Wallis test, nC. elegans = 17–34) and (B) nlp-49 strains (one-way ANOVA, nC. elegans = 15–18) during 1-hour predator. (C, D) Latency to exit the patch following a bite-induced escape response, averaged across escape-induced exits, for (C) seb-3 strains (Kruskal–Wallis test, nC. elegans = 17–34) and (D) nlp-49 strains (Kruskal–Wallis test, nC. elegans = 14–18). Error bars are 95% bootstrap CIs containing the mean. n.s. = p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Outstretched feeding on high-density bacteria by nlp-49 strains.
Percentage of animals adopting outstretched feeding posture in nlp-49 deletion and overexpression strains (Fisher’s exact test with Benjamini–Hochberg adjustment, C. elegans = 35–54).
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Figure 3—figure supplement 3—source data 1
Data for Figure 3—figure supplement 3.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig3-figsupp3-data1-v1.zip

New patch entry latency by seb-3 and nlp-49 strains.
(A, B) Latency to enter a new patch for (A) seb-3 strains (nC. elegans = 16–35) and (B) nlp-49 strains (nC. elegans = 14–22) (Wilcoxon’s ranked sum test with Benjamini–Hochberg adjustment). (C, D) Comparison of latency to enter a new patch, within predator or predator-free conditions for (C) seb-3 strains (Kruskal–Wallis test, nC. elegans = 16–35) and (D) nlp-49 strains (Dunn’s test with Benjamini–Hochberg adjustment, nC. elegans = 14–22). Error bars are 95% bootstrap CIs containing the mean. n.s. = p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
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Figure 3—figure supplement 4—source data 1
Data for Figure 3—figure supplement 4A and C.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig3-figsupp4-data1-v1.zip
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Figure 3—figure supplement 4—source data 2
Data for Figure 3—figure supplement 4B and D.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig3-figsupp4-data2-v1.zip

Interdependence between SEB-3 and NLP-49 peptides shifts across defense modes.
(A) Probability of exit following an escape response by seb-3 deletion strains with nlp-49 deletion or overexpression (binomial logistic regression followed by Wald test with single-step adjustment for Tukey contrasts, nC. elegans = 11–24). (B) Latency to reenter the patch following an exit, averaged across escape-induced exits, for strains with seb-3 deletion by itself or with nlp-49 deletion or overexpression (Games–Howell test, nC. elegans = 11–21). (C) Percentage of animals adopting outstretched feeding posture in seb-3 deletion strains with nlp-49 deletion or overexpression (Fisher’s exact test with Benjamini–Hochberg adjustment, nC. elegans = 67–78). Significance asterisks represent comparisons with seb-3(-);nlp-49(-) (blue). (D) Percentage of animals adopting outstretched feeding posture in seb-3 deletion strains with nlp-49 deletion or overexpression (Fisher’s exact test with Benjamini–Hochberg adjustment, nC. elegans = 64–67). Significance asterisks represent comparisons with seb-3(-);nlp-49(o/e) (yellow). (E) Bins explored following 4-hour exposure to predator or predator-free conditions, by seb-3 deletion strains with nlp-49 deletion or overexpression (Student’s t-test with Benjamini-Hochberg adjustment, nC. elegans = 9–10). (F) Bins explored following 4-hour exposure to predator or predator-free conditions, by seb-3 deletions strains with or without nlp-49 overexpression (Student’s t-test with Benjamini–Hochberg adjustment, nC. elegans = 13–16). Error bars in (A) are predicted P(exit | escape) and 95% CIs from binomial logistic regression model of data. All other error bars are 95% bootstrap CIs containing the mean. n.s. = p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
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Figure 4—source data 1
Data for Figure 4A, Figure 4—figure supplement 1A and B.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig4-data1-v1.zip
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Figure 4—source data 2
Data for Figure 4B.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig4-data2-v1.zip
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Figure 4—source data 3
Data for Figure 4C.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig4-data3-v1.zip
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Figure 4—source data 4
Data for Figure 4D.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig4-data4-v1.zip
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Figure 4—source data 5
Data for Figure 4E.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig4-data5-v1.zip
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Figure 4—source data 6
Data for Figure 4F.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig4-data6-v1.zip

Circa-strike behavior of seb-3 deletion strains with nlp-49 deletion or over expression.
(A) Number of bite-induced escape responses (one-way ANOVA, nC. elegans = 11–24). (B) Latency to exit the patch following a bite-induced escape response,averaged across escape-induced exits (Tukey’s test, nC. elegans = 11–24). Error bars are 95% bootstrap CIs containing the mean.n.s. = p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Baseline locomotion of seb-3 deletion strains with nlp-49 deletion or overexpression.
(A) Baseline speed in bacteria-free, predator-free conditions (one-way ANOVA, nC. elegans = 10). (B) Latency to enter a new patch for mock-exposed double deletion mutants and wildtype animals (Dunn’s test, nC. elegans = 9–10). Error bars are 95% bootstrap CIs containing the mean. n.s. = p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
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Figure 4—figure supplement 2—source data 1
Data for Figure 4—figure supplement 2B.
- https://cdn.elifesciences.org/articles/98262/elife-98262-fig4-figsupp2-data1-v1.zip
Tables
C. elegans and Pristionchus spp. strains.
Strain Name | Source | Genotype |
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N2 | CGC | Wildtype |
RS5194 | Click et al., 2009 | P. pacificus wild isolate |
PS312 | Click et al., 2009 | P. pacificus wild isolate |
JU1051 | Félix et al., 2013 | P. uniformis wild isolate |
TU445 | Ragsdale et al., 2013 | P. pacificus eud-1(tu445) |
IV820 | This study | seb-3(tm1848) X outcrossed 4x |
IV496 | This study | seb-3(tm1848) X; ueEx309[Pseb-3::seb-3-GFP] |
AQ3644 | Chew et al., 2018 | nlp-49(gk546875)X |
AQ3853 | Chew et al., 2018 | nlp-49(gk546875)X; ljEx1004[Pnlp-49::Pnlp-49gDNA+ UTR::SL2-mKate2(25); unc-122::gfp(50)] |
AQ3701 | Chew et al., 2018 | seb-3(tm1848); nlp-49(gk546875) |
AQ3851 | Chew et al., 2018 | seb-3(tm1848); ljEx1004[Pnlp-49::Pnlp-49 gDNA+ UTR::SL2-mKate2(25); unc-122::GFP(50)] |
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Strains are C. elegans unless otherwise indicated.
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CGC, Caenorhabditis Genetics Center.