Reef Sound as an Orientation Cue for Shoreward Migration by Pueruli of the Rock Lobster, <i>Jasus edwardsii</i>

<div><p>The post-larval or puerulus stage of spiny, or rock, lobsters (Palinuridae) swim many kilometres from open oceans into coastal waters where they subsequently settle. The orientation cues used by the puerulus for this migration are unclear, but are presumed to be critical to finding a place to settle. Understanding this process may help explain the biological processes of dispersal and settlement, and be useful for developing realistic dispersal models. In this study, we examined the use of reef sound as an orientation cue by the puerulus stage of the southern rock lobster, <i>Jasus edwardsii</i>. Experiments were conducted using <i>in situ</i> binary choice chambers together with replayed recording of underwater reef sound. The experiment was conducted in a sandy lagoon under varying wind conditions. A significant proportion of puerulus (69%) swam towards the reef sound in calm wind conditions. However, in windy conditions (>25 m s^-1) the orientation behaviour appeared to be less consistent with the inclusion of these results, reducing the overall proportion of pueruli that swam towards the reef sound (59.3%). These results resolve previous speculation that underwater reef sound is used as an orientation cue in the shoreward migration of the puerulus of spiny lobsters, and suggest that sea surface winds may moderate the ability of migrating pueruli to use this cue to locate coastal reef habitat to settle. Underwater sound may increase the chance of successful settlement and survival of this valuable species.</p></div

Map of the experimental site.

<p>Castle Point experimental sites used for collection of <i>Jasus edwardsii</i> pueruli and experimentation with behavioural choice chambers. “X” = positions of underwater speakers. The coastline was extracted from <a href="https://www.ngdc.noaa.gov/mgg/shorelines/" target="_blank">https://www.ngdc.noaa.gov/mgg/shorelines/</a> and redrawn.</p

Underwater sounds at the experimental site.

<p>Power spectra of underwater sound recorded for each night at the experimental site in January 2015 (A) immediately prior to the artificial source of underwater sound commencing (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157862#pone.0157862.s002" target="_blank">S2 Sound File</a>), and (B) during the experiments with the artificial source of underwater sound present (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157862#pone.0157862.s003" target="_blank">S3 Sound File</a>).</p

Experimental set up and choice chamber.

<p>(A) Schematic of experimental set up, and (B) behavioural choice chamber design.</p

Directional choice of <i>Jasus edwardsii</i> pueruli in the behavioural choice chamber (towards or away from the artificial sound), position of the speaker, and ambient environmental variables at the experimental site (timing of low tide, gust wind direction and speed).

<p>Directional choice of <i>Jasus edwardsii</i> pueruli in the behavioural choice chamber (towards or away from the artificial sound), position of the speaker, and ambient environmental variables at the experimental site (timing of low tide, gust wind direction and speed).</p

Predicted probability and observed proportion of puerulus choosing to move towards the reef sound for the range of wind gust speeds experienced over the study period.

<p>Predicted probability of a <i>Jasus edwardsii</i> puerulus choosing to move towards the reef sound moderated by the wind gust speed (m s^-1), based on a logistic regression (grey area represents the 90% CI) (Wald Z = -2.4; P = 0.02; n = 59). Black dots in the graph represent the observed proportion of the puerulus per night choosing to move towards the reef sound, while the numbers adjacent to the black dots represents the total puerulus sample size used to generate the proportion.</p

Logistic regression of the directional choice of <i>Jasus edwardsii</i> pueruli in relation to the speaker position and environmental variables (tide phase, wind gust direction and wind gust speed).

<p>Logistic regression of the directional choice of <i>Jasus edwardsii</i> pueruli in relation to the speaker position and environmental variables (tide phase, wind gust direction and wind gust speed).</p

TCR/CD3- and CD59-mediated Ca²⁺ signaling are dependent on Lck and LAT.

<p>Cluster distribution of Ca²⁺ time traces in differently treated cells upon anti-CD3 and anti-CD59 stimulation. Each color represents the percentage of a certain Ca²⁺ time trace cluster in the cell population. (A) Ca²⁺ measurements were performed with WT cells transiently transfected with negative control siRNA (siNeg), Lck-specific siRNA (siLck), WT cells treated with 10 µM PP2 (PP2), and Lck-deficient J.CaM1.6 cells. Clusters representing Ca²⁺ release patterns are framed in black (89.4±10.1%, 76.5±10.4%, 28.3±39.1%, and 23.4±9.9% upon anti-CD3 stimulation, 36.9±13.2%, 12.7±6.1%, 2.6±2.0%, and 1.2±1.2% upon anti-CD59 stimulation for siNeg, siLck, PP2-treated, and J.CaM1.6 cells, respectively). Mean values from at least two independent experiments, each with three technical replicates, are shown (n ≥ 204 per cell type and condition). (B) Cluster analysis of Ca²⁺ time traces in WT cells transiently transfected with negative control siRNA (siNeg), LAT-specific siRNA (siLAT), and LAT-deficient J.CaM2.5 cells. Clusters representing Ca²⁺ release patterns are framed in black (89.4±10.1%, 73.5±5.8%, and 3.0±2.3% upon anti-CD3 stimulation, 36.9±13.2%, 13.0±6.6%, and 1.7±1.6% upon anti-CD59 stimulation for siNeg, siLAT, and J.CaM2.5 cells, respectively). Mean values from at least three independent experiments, each with three technical replicates, are shown (n ≥ 208 per cell type and condition). Multiple comparison tests for the fractions showing Ca²⁺ release patterns in (A) and (B) were assessed by one-way ANOVA, significances are shown where applicable, ** p < 0.01, *** p < 0.001. (C) Knock-down of target proteins was tested by Western blotting. 48 h after transfection cell lysates from siRNA treated cells were probed with anti-Lck, anti-LAT, and anti-β-actin. J.CaM1.6 cells, J.CaM2.5 cells, and WT cells served as controls.</p

CD59-mediated Ca²⁺ signaling requires CD3ζ expression.

<p>(A) Cluster distribution of Ca²⁺ time traces in WT and TCR^high cells is shown upon anti-CD3 and anti-CD59 stimulation. Each color represents the percentage of a certain Ca²⁺ time trace cluster in the cell population. Clusters representing Ca²⁺ release patterns are framed in black (91.4±2.1% and 92.9±3.9% upon anti-CD3 stimulation, 34.4±16.3% and 72.0±16.6% upon anti-CD59 stimulation in WT and TCR^high cells, respectively). Mean values from two independent experiments, each with three technical replicates are shown (n ≥ 167 per cell type and condition). (B) Cluster distribution of Ca²⁺ time traces in WT, TCR^-, and cells expressing CD8-ζ fusion protein is shown upon anti-CD3 and anti-CD59 stimulation. Each color represents the percentage of a certain Ca²⁺ time trace cluster in the cell population. Clusters representing Ca²⁺ release patterns are framed in black (90.1±1.4%, 1.9±0.7% and 7.4±2.3% upon anti-CD3 stimulation, 25.2±6.8%, 1.6±0.4% and 13.4±1.6% upon anti-CD59 stimulation for WT, TCR^-, and CD8-ζ cells, respectively). Mean values from four independent experiments, each with three technical replicates, are shown (n ≥ 343 per cell type and condition). Multiple comparison tests for the fractions showing Ca²⁺ release patterns in (A) and (B) were assessed by one-way ANOVA, significances are shown where applicable, * p < 0.05, ** p < 0.01, *** p < 0.001.</p

Reconstitution of Lck by forced interaction of CD3ζ and Lck facilitates TCR/CD3- but not CD59-mediated Ca²⁺ signaling.

<p>(A) Lck expression levels in WT cells, J.CaM1.6 cells and J.CaM1.6 cells expressing mEGFP-tagged Lck fused to CD3ζ (CD3ζ-Lck) were tested by Western blotting, the same blot was reprobed using anti-β-actin as a control. (B) Plasma membrane localization of CD3ζ-Lck-mEGFP in J.CaM1.6 cells was imaged by fluorescence microscopy. CD3ζ-Lck-mEGFP fluorescence and a bright field (BF) image of a transfected J.CaM1.6 cell are shown (scale bars  =  10 µm). (C) Cluster distribution of Ca²⁺ time traces in Lck-deficient J.CaM1.6 cells and J.CaM1.6 cells stably expressing mEGFP-tagged Lck fused to CD3ζ (CD3ζ-Lck) is shown upon anti-CD3 or anti-CD59 stimulation. Each color represents the percentage of a certain Ca²⁺ time trace cluster in the cell population. Clusters representing Ca²⁺ release patterns are framed in black (35.7±4.9% and 71.8±2.3% upon anti-CD3 stimulation, 1.4±2.3% and 1.0±1.3% upon anti-CD59 stimulation for J.CaM1.6 and CD3ζ-Lck cells, respectively). Mean values from three independent experiments, each with three technical replicates, are shown (n ≥ 323 per cell type and condition). Multiple comparison tests for the fractions showing Ca²⁺ release patterns were assessed by one-way ANOVA, significances are shown where applicable, ***p < 0.001.</p