The power spectrum of the complex click stimulus used to identify the size/age for the onset of the acoustically evoked behavioral response.

<p>The majority of the energy in the stimulus is located below 700 µPa or −15.2 dB re 1 g in the Z-axis of stimulation. Juvenile and adult midshipman have greatest auditory sensitivity at frequencies below 300 Hz.</p

The acoustically evoked behavioral response (AEBR) of fish to a complex click stimulus with a peak SPL of 154 dB re 1 µPa or −15.2 dB re 1 g in the Z-axis of stimulation.

<p>The AEBRs are shown as the percentage of the tested fish (60 midshipman larvae and 2 juveniles) that responded to the stimulus. Note that none of the small midshipman larvae less than 1.4 cm TL responded to the stimulus, whereas all of the midshipman larvae greater than 1.8 cm TL responded. Thus onset of the acoustically evoked behavioral response is estimated to occur between 1.4–1.8 cm TL. The solid line represents a best-fit sigmoidal curve.</p

Development of the Acoustically Evoked Behavioral Response in Larval Plainfin Midshipman Fish, <i>Porichthys notatus</i>

<div><p>The ontogeny of hearing in fishes has become a major interest among bioacoustics researchers studying fish behavior and sensory ecology. Most fish begin to detect acoustic stimuli during the larval stage which can be important for navigation, predator avoidance and settlement, however relatively little is known about the hearing capabilities of larval fishes. We characterized the acoustically evoked behavioral response (AEBR) in the plainfin midshipman fish, <i>Porichthys notatus</i>, and used this innate startle-like response to characterize this species' auditory capability during larval development. Age and size of larval midshipman were highly correlated (r² = 0.92). The AEBR was first observed in larvae at 1.4 cm TL. At a size ≥1.8 cm TL, all larvae responded to a broadband stimulus of 154 dB re1 µPa or −15.2 dB re 1 g (z-axis). Lowest AEBR thresholds were 140–150 dB re 1 µPa or −33 to −23 dB re 1 g for frequencies below 225 Hz. Larval fish with size ranges of 1.9–2.4 cm TL had significantly lower best evoked frequencies than the other tested size groups. We also investigated the development of the lateral line organ and its function in mediating the AEBR. The lateral line organ is likely involved in mediating the AEBR but not necessary to evoke the startle-like response. The midshipman auditory and lateral line systems are functional during early development when the larvae are in the nest and the auditory system appears to have similar tuning characteristics throughout all life history stages.</p></div

Best evoked frequency (BEF) histograms of the acoustically evoked behavioral response in midshipman larvae based on sound pressure level (SPL, black bars) and particle acceleration (gray bars).

<p>The distribution of the BEF for the AEBR is based on the individual AEBR profiles for all the midshipman larval groups tested. Note that the BEF is defined as the frequency with the lowest threshold to evoke the AEBR).</p

The relationship between particle motion (acceleration) and sound pressure in the experimental tank used to test the AEBR in midshipman fish.

<p>Particle motion was measured using a 3D accelerometer after calibrating stimulus frequencies using sound pressure such that all stimulus frequencies at a peak SPL within 2 µPa. Here we display the particle motion measured in the X-, Y-, and Z-axes for all test frequencies and intensities. Note that the Z-axis represents the main axis of stimulation.</p

Acoustically evoked behavioral response (AEBR) profiles for the four size groups of midshipman larvae (small, medium, large) and juveniles.

<p>The top portion of the graphs shows the response profiles in terms of SPL and the bottom portion of each graph is displayed in terms of acceleration (particle motion) in the Z (vertical)-axis of stimulation. Small midshipman larvae (A) are depicted by the line with solid circles, medium midshipman larvae (B) with open circles, large midshipman larvae (C) with solid triangles, and the juveniles (D) with open triangles. Over all the response profiles for all four groups were similar in shape with greatest sensitivity at the lowest test frequencies (<225 Hz).</p

The presence of mechanosensory neuromasts as a function of fish total length (TL) in midshipman larvae.

<p>Mechanosensory neuromasts are shown as the percentage of fish examined that had neuromasts present. The presence of mechanosensory neuromasts was determined by the uptake of the vital dye DASPEI, which is taken up by energetically active cells such as lateral line neuromasts and can be visualized <i>in vivo</i>. Fish were scored base on DASPEI staining in a binary fashion (yes/no): yes, for the staining of one or more neuromasts and no, for a lack of neuromast cell staining. Note that none of the small midshipman larvae less than 1.6 cm TL had any detectable neuromast cells, whereas all larvae greater than 1.8 cm TL had at least one neuromast cell with DASPEI staining. The solid line represents a best-fit sigmoidal curve.</p

Video frame sequence of a representative acoustically evoked behavioral response (AEBR).

<p>The images show a 2.1–H show the fish positions during and after stimulus presentation. Note that in image C the caudal fin is curved toward the head of the fish, almost forming a C shape. A positive AEBR was only considered when the caudal fin moved greater than ½ of the fish's total length directly following a stimulus presentation.</p

Differences in neuromast number across groups.

<p>(A) SN were clustered in one of six discrete ‘stitches’ or groupings, which we term for convenience in the present report S1–S6. Each of these stitches was treated as a region of interest for analysis of neuromast number. (B–E) Examples of DASPEI-labeled neuromasts from wild-origin juveniles (B–C) or Cook Creek hatchery fish (D–E). (B) Stitches S1–S2, showing how S2 intersects S1 near its midpoint, running anteroventrally toward the eye. Scale bar = 1 mm. (C) Low-magnification image of the left side of the head, showing the stitch around the naris (S3) and the stitch over the operculum (S4). The infraorbital canal (IC) is also labeled in this image. This canal was not clearly visible in all specimens so neuromast number was not quantified. Scale bar = 2 mm. (D) Stitch S3 (bordering a right-side naris) under higher magnification. Scale bar = 1 mm. (E) SN from stitch S5 (arrowheads), which sits atop the trunk canal (arrows). Trunk canal neuromasts are elongated in the rostrocaudal direction, while adjacent SN are oriented dorsoventrally. Scale bar = 1 mm. (F) Total neuromast number (summed across left and right sides) per fish (open circles) and per group (filled circles, mean±1 SEM, <i>n</i> = 10 fish per group). There were significant differences in neuromast number between groups (one-way ANOVA <i>F</i>_1,2 = 9.45, <i>p</i> = 0.001). (G) SN number comparisons within each ROI using one-way ANOVA followed by Tukey's post-hoc analysis. Individual and group data are plotted across ROIs. Statistical tests are summarized are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059162#pone-0059162-t001" target="_blank">Table 1</a>.</p

High variability in hair cell number within neuromasts.

<p>(A–C) Confocal images (brightest-point projections) of neuromasts double-labeled with anti-acetylated tubulin (red) and phalloidin (green), showing the kinocilia and hair bundles/cuticular plates, respectively. The phalloidin label also delineates overall neuromast architecture. (A) Three neuromasts from stitch S2 of a wild-origin fish, showing neuromast morphology and spacing. (B) Single SN from stitch S3 of a Lake Quinault hatchery fish, demonstrating the rounded morphology sometimes observed. In contrast, elongated SN were more typically noted, illustrated here by the S5 neuromast from a Cook Creek fish (C). (D) Average hair cell number for 4 randomly selected individuals (open circles) from each group for which complete data (SN in all four dissected ROIs S1–S4; range 3–21 SN per ROI) were available, and group means (filled circles, mean ± 1 SEM) for each ROI. There were no significant cross-group differences in hair cell number (<i>p</i>>0.05).</p