A sensation for inflation: initial swim bladder inflation in larval zebrafish is mediated by the mechanosensory lateral line

Larval zebrafish achieve neutral buoyancy by swimming up to the surface and taking in air through their mouths to inflate their swim bladders. We define this behavior as ‘surfacing’. Little is known about the sensory basis for this underappreciated behavior of larval fish. A strong candidate is the mechanosensory lateral line, a hair cell-based sensory system that detects hydrodynamic information from sources like water currents, predators, prey, and surface waves. However, a role for the lateral line in mediating initial inflation of the swim bladder has not been reported. To explore the connection between the lateral line and surfacing, we utilized a genetic mutant (lhfpl5b−/−) that renders the zebrafish lateral line insensitive to mechanical stimuli. We observe that approximately half of these lateral line mutants over-inflate their swim bladders during initial inflation and become positively buoyant. Thus, we hypothesize that larval zebrafish use their lateral line to moderate interactions with the air-water interface during surfacing to regulate swim bladder inflation. To test the hypothesis that lateral line defects are responsible for swim bladder over-inflation, we show exogenous air is required for the hyperinflation phenotype and transgenic rescue of hair cell function restores normal inflation. We also find that chemical ablation of anterior lateral line hair cells in wild type larvae causes hyperinflation. Furthermore, we show that manipulation of lateral line sensory information results in abnormal inflation. Finally, we report spatial and temporal differences in the surfacing behavior between wild type and lateral line mutant larvae. In summary, we propose a novel sensory basis for achieving neutral buoyancy where larval zebrafish use their lateral line to sense the air-water interface and regulate initial swim bladder inflation.


Introduction
The swim bladder of teleost fish provides for neutral buoyancy in the water column, thereby minimizing the energy expenditure associated with locomotion (Alexander, 1966). Larval teleosts initate swim bladder inflation by swimming up to the surface to ingest air using a set of behaviors we refer to as "surfacing" (Evans and Damant, 1928;Lindsey et al., 2010;von Ledebur, 1928).
Although surfacing poses a significant risk of predation, larval fish are highly motivated to reach the surface to intake air. For example, pre-inflated lake trout larvae will swim hundreds of feet at a constant rate in attempts to access the surface (Tait, 1960). Surfacing is a complex task that requires a combination of newly developed physical features, sensory systems, and motor skills (Bailey and Doroshov, 1995;Chatain, 1989;Doroshev et al., 1981;Kimmel et al., 1995;Kitajima et al., 1994) -(i) Larvae must discriminate up from down and (ii) be able to move directionally towards the surface; (iii) they must appropriately sense the air-water interface, and (iv) be able to intake air through the mouth upon arrival at the surface. Ingested air is then moved by peristalsis into the swim bladder through the pneumatic duct that connects the gut and swim bladder (Doroshev et al., 1981;Rieger and Summerfelt, 1998;Tait, 1960). Together, surfacing represents perhaps the most complex behavior that larvae perform prior to achieving neutral buoyancy (Fero et al., 2011).
Little is known about the sensory cues that instruct the surfacing behavior. Perturbations to the vestibular system typically result in hypoinflation (Nicolson et al., 1998;Schoppik et al., 2017).
The inability to detect gravity by the utricular otolith is specifically responsible for the phenotype, presumably because larvae are unable to orient their movements upwards towards the surface (Riley and Moorman, 2000). Photosensory cues may also play a role in directional orientation and surface detection. However, which photosensory systems are involved has not been defined and the effects of altering lighting conditions varies between species (Stuart and Drawbridge, 2012;Trotter et al., 2003;Villamizar et al., 2009). Even less is known about how larval fish detect the air-water interface itself. Fish and some amphibians possess an additional hair cell-based sensory system -the mechanosensory lateral line -that contributes to detection of near-field hydrodynamic information originating from water currents, predators and prey, conspecifics, abiotic objects, and surface waves (Anneser et al., 2020;Bleckmann, 2006;Butler and Maruska, 2016;Carrillo and McHenry, 2016;Mogdans, 2019;Montgomery et al., 1997;Stewart et al., 2013;Venuto et al., 2022). Specific to surface detection, surface-feeding fish use their lateral line to detect surface waves created by their prey (Bleckmann and Schwartz, 1982). Furthermore, Japanese flying fish are predicted to use their ventrally-located lateral line to sense transitions through the air-water interface (Gibbs, 2004;Tsukamoto and Yoshino, 1957). While the lateral line is a strong candidate to aid in surfacing behaviors that lead to initial swim bladder inflation in larval fish, its specific role in this important behavior has not been explored.
A CRISPR-Cas9 knockout of LHFPL tetraspan subfamily member 5b (lhfpl5b) is the first genetic zebrafish mutant where the lateral line is non-functional from birth but hearing and balance are normal (Erickson et al., 2020). These mutants provide an opportunity to uncover novel roles for the lateral line. Unexpectedly, homozygous mutant larvae present with a hyperinflation phenotype characterized by over-filled swim bladders and positive buoyancy, a very rare finding among swim bladder defects (Abbas and Whitfield, 2009;McCune and Carlson, 2004). Using several genetic and chemical techniques to manipulate lateral line function, we show that lateral line sensory information is used by larval zebrafish to appropriately fill their swim bladders, likely by detecting surface tension at the air-water interface using mechanosensory neuromasts located on their heads. Overall, our study uncovers a novel role for the mechanosensory lateral line mediating initial inflation of the swim bladder.

Animal husbandry and ethics statement
Adult zebrafish (Danio rerio) were maintained and bred using standard procedures (Westerfield, 2000). All experiments used larvae at 2-6 days post-fertilization (dpf), which are of indeterminate sex at this stage. Except where otherwise stated, animals were placed in clear, plastic tubs (dimensions: 15 cm x 10 cm x 4 cm, E3 volume: 460 mL) at 2 dpf and maintained at 28.5°C on a 14:10 light/dark cycle at 500-700 lux in E3 embryo media (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgCl2, buffered with NaHCO3). Unhatched embryos were manually dechorionated before placing larvae in experimental conditions at 2 dpf. It is worth noting that there was no obvious difference in wild type versus lhfpl5b mutant hatching rate, though this was not explicitly studied. Animal research complied with guidelines stipulated by the Institutional Animal Care and Use Committees at East Carolina University (protocol D372) and Amherst College (assurance number 3925-1 with the Office of Laboratory Animal Welfare).

Mutant and transgenic fish lines
The zebrafish mutant allele lhfpl5b vo35 and transgenic lines Tg(myo6b:EGFP-lhfpl5a)vo23 and Tg(myo6b:EGFP-PA)vo68 were used in this study (Erickson et al., 2017;Erickson et al., 2020). For all experiments, lhfpl5b vo35 homozygotes were identified from their hetero-and homozygous wild type siblings by lack of FM 1-43 (ThermoFisher) labeling of the lateral line hair cells.
To create a fish line that expresses the Channelrhodopsin-2 (ChR2) optogenetic protein solely in the hair cells of the lateral line, approximately 2 kb of DNA upstream of the start codon of the lhfpl5b gene was cloned into the p5E plasmid of the recombination-based multisite Gateway cloning system (ThermoFisher). A Tol2 transposon backbone was then used to create the injectable expression plasmid lhfpl5b2028:ChR2-EYFP-PA. This plasmid was then co-injected with transposase mRNA into single-cell embryos (Kwan et al., 2007) to create the Tg(lhfpl5b2028:ChR2-EYFP-PA) zebrafish line.

Buoyancy tests
To ascertain the buoyancy status of larval fish, larvae were anesthetized with MS-222 (Western Chemical Inc., Ferndale, WA) and placed at the vertical midway point of a 50 mL conical tube containing E3 media. At the expiration of a 30-second timer, the end position of the fish was recorded and the fish was removed from the tube for imaging under the microscope. If the fish reached the top or bottom of the tube before 30 seconds, then the time it took to reach that location was recorded. The total height of the water column was 8.9 cm, and the fish was placed half-way at 4.45 cm. The rate of buoyancy was determined by dividing the centimeters travelled by the seconds it took to travel that distance (cm/s). Over-inflated fish were categorized by larvae floating to the top of the tube in 30 seconds, while under-inflated fish sunk to the bottom of the tube.
Neutrally boutant or regular-inflated larvae remained near the middle of the water column. This test was conducted during the imaging portion at the end of each experiment, with the exception of the oil-water interface experiment where buoyancy could not be accurately determined due to the greater density of oil compared to air.

Imaging and swim bladder measurements
Swim bladders were imaged using a SteREO Discovery.V8 microscope (Zeiss) equipped with a Gryphax Arktur camera (Jenoptik). Lateral and dorsal images of the swim bladder were taken for each fish. Swim bladder measurements were conducted using the measurement tool in Adobe Photoshop where measurements were calibrated according to the image of the stage micrometer taken when imaging experiments. The major axis and minor axis of the lateral view of the swim bladder was measured as well as the minor axis of the dorsal view. All measurements were taken from numbered images so that the measurer was blinded to the genotype/condition. These meaurements were halved and used in the equation for the volume of an ellipsoid: = 4/3 to find the volume of the swim bladder (Lindsey et al., 2010).

Manipulation of photosensory cues during surfacing
At 0 dpf, zebrafish embryos were collected and immediately placed in plastic tubs filled with E3 (detailed above) in an unlit incubator. Larvae were not brought into light until 6 dpf, at which point lateral line mutants and wild type siblings were identified by FM 1-43 labeling. After sorting, larvae were analyzed for buoyancy. The experiment was repeated three times using 20-21 larvae (combined mutant and wild type) per trial.

Blocking access to the air-water interface
Mutant larvae and wild type siblings were sorted at 2 dpf into glass cylinders (diameter: 9.5 cm, E3 volume: 425 mL) equipped with fitted, wire mesh filter plungers (Bodum Inc., USA).
Conditions were as follows: wild type with open access to the surface, wild type with blocked access to the surface, mutant with open access to the surface, and mutant with blocked access to the surface. All containers were filled half way with E3 and in the experimental condition ("blocked access"), the filter was pressed down below the surface for both wild type and mutant larvae. The control condition used the same amount of E3 but the filter remained above the surface so that larvae could access the exogenous air. Conditions remained constant until 6 dpf when larvae were imaged and analyzed for swim bladder inflation. The experiment was repeated three times using 15-25 larvae per condition.

Analysis of surfacing behavior
Wild type and lhfpl5b vo35 larvae were were sorted at 2 dpf and blocked from having access to the surface until 9 AM on 4 dpf when individuals were immediately placed into clear rectangular containers (dimensions: 3 cm x 3 cm x 3 cm, E3 volume: 20 mL) for video recording. Recordings lasted 12 hours, from 9 AM to 9 PM on 4 dpf. At the end of filming, larvae were imaged and analyzed for buoyancy and swim bladder volume, as described. All surfacing videos were numbered for blind analysis. Surfacing analysis was conducted by manual observation of each individual larvae from the videos (30 frames per second) and manually counting visits made to the surface (counted as reaching the meniscus), the timeframe of the video at which the surfacing event occurred, and the duration of time spent at the surface per surfacing event. Recording was repeated six times with both mutant wild type larvae present in each recording. Larvae that did not inflate their swim bladders by the end of filming (5 total larvae, 1 wild type and 4 mutants) were excluded from this analysis due to lack of surfacing events.

Lateral line hair cell ablations
For all experiments, wild type and lhfpl5b mutant larvae were treated with ototoxin with untreated siblings acting as controls (n = 7-15 larvae per condition per trial, 4 trials) For single treatments, surface accessed was blocked as described above for untreated and treated larvae until 9 AM on 4 dpf when treatment occurred. Single 30 minute treatments with neomycin sulfate (EMD Millipore Corp., USA) were done with a 100 µm solution in E3 while repeated neomycin treatments (Venuto and Erickson, 2021) were done as previously described with a concentration of 50 µM. For full ablation with copper sulfate (CuSO4; Ward's Science, Rochester, NY), the treatment concentration was 10 uM with a 30 minute exposure. For experiments where head and trunk neuromasts were selectively ablated with CuSO4, anesthetized larvae were moved to a rectangular plate (4.5 cm x 3 cm x 1 cm) coated with Sylgard (Electron Microscopy Sciences, Hatfield, PA). After removing excess water, a thin layer of petroleum jelly (Vaseline, Unilever USA) was placed horizontally at the back of the head. Next, 30 uM CuSO4 or E3 was pipetted onto the appropriate region depending on the experimental condition. Immediately following the 13 minute treatment, larvae were placed in fresh E3 in clear tubs detailed above for 24 hours until 9 AM on 5 dpf when larvae were imaged and analyzed for swim bladder inflation. For all experiments, larvae were imaged and analyzed for swim bladder inflation at 9 AM on 5 dpf. Each experiment was repeated three times using 10-24 larvae per condition.

Transgenic rescue of lateral line mutant
Larval zebrafish from crosses of lhfpl5b +/and Tg(myo6b:EGFP-lhfpl5a)vo23Tg; lhfpl5b +/were sorted at 2 dpf for transgene expression. At 6 dpf, larvae were numbered, imaged for swim bladder inflation and genotyped for the lhfpl5b vo35 allele as previously described (Erickson et al., 2020). The experiment was repeated twice using 26-48 larvae per condition.

Optogenetic activation of lateral line hair cells with Channelrhodopsin-2 (ChR2)
After blocking access to the surface until 4 dpf, we exposed wild type and lateral line mutants, with or without the lhfpl5b2028:ChR2-EYFP-PA transgene, to LED blue light (470 nm) with an intensity of 730 lux over their holding tanks against a dark background. Light flashed for 25 milliseconds at one second intervals. Control larvae were kept in the same background, without blue light exposure. Conditions were held constant until 6 dpf when larvae were sorted for genotype, imaged and analyzed for swim bladder inflation. The experiment was repeated three times using 15-25 larvae per condition.

Oil-water interface experiments
Mutant larvae and wild type siblings were sorted at 2 dpf into clear cylindrical containers (diameter: 5 cm , volume: 60 mL) with embryo media only or embryo media with a 2 mm thick surface layer of lab grade mineral oil (Ward's Science, Rochester, NY). Oil on the surface decreases surface tension (Johansen, 1924) and has been previously used in experiments involving the zebrafish swim bladder (Ehrlich and Schoppik, 2017). Conditions remained constant until 6 dpf when larvae were imaged and analyzed for swim bladder volume. Because oil-filled larvae are negatively buoyant, we did not perform buoyancy tests and classified larvae as over-filled based on swim bladder volume alone. The experiment was repeated three times using 15-25 larvae per condition.

Graphs and statistical tests
All graphs and statistical tests were done using R (R Core Team, 2022). One-way ANOVAs were used to compare the conditions as a whole (genotype independent variable plus environment independent variable) with Tukey post-hoc test. Chi-squared tests were used to analyze proportion data for over and under-inflation. For the over-inflation comparisons, the two categories used in the comparison between conditions/genotypes were (i) over-inflation and (ii) all other inflation (regular and under), meaning that totals per category/genotype accounted for 100% of the population. Similarly, for the under-inflation comparisons, the two categories used in the comparison between conditions/genotypes were (i) under-inflation and (ii) all other inflation (regular and over). P-values less than 0.05 were considered significant.

Lateral line mutants exhibit a swim bladder hyperinflation phenotype
The lateral line has not been previously implicated in initial inflation of the larval swim bladder. As such, it was unexpected to observe that lateral line mutants (lhfpl5b -/-) exhibit hyperinflated swim bladders by 5-6 dpf ( Figure 1A). Based on our swim bladder volume and buoyancy measurements, we categorized each larva in the mutant (n = 31) and wild type ( This unexpected hyperinflation phenotype in lhfpl5b vo35 homozygotes led us to investigate if the lateral line is contributing important sensory information during the surfacing behavior.

Access to the air-water interface is required for the hyperinflation phenotype of lateral line mutants
For most teleosts, initial swim bladder inflation requires an intake of exogenous air, typically from the water's surface. To test if the hyperinflation phenotype of lateral line mutants requires access to surface air, we physically blocked access to the surface from 2-6 dpf ( Figure   2A). As expected for wild type larvae, blocking access to the surface led to significantly more underinflated larvae ( Figure 2B) and a significant decrease in the average swim bladder volume post-test, p = 0.972). From these data, we concluded that the lhfpl5b mutant hyperinflation phenotype requires intake of exogenous air and is not caused by abnormal gas exchange from surrounding media nor excess internal gas production.

Transgenic rescue of lateral line mutants restores normal inflation
We next examined if lateral line defects are responsible for the hyper-inflation phenotype of lhfpl5b mutants by restoring lateral line function in mutant larvae. We used the Tg(myo6b:EGFP-lhfpl5a)vo23 transgene, which uses a hair cell specific promoter (myo6b) to drive a GFP-tagged lhfpl5a gene (Erickson et al., 2017). It has been shown that this lhfpl5a transgene rescues lateral line hair cell function in lhfpl5b -/-(or lateral line) mutant fish (Erickson et al., 2020).
Because the restoration of lhfpl5 function specifically in hair cells mitigates hyperinflation in lhfpl5b mutants, we concluded that the phenotype is due to defects in sensory hair cell function and not caused by an unrecognized role for lhfpl5b in another sensory organ or the swim bladder itself.

Selective ablation of head neuromasts produces a hyperinflation phenotype in wild type larvae
To further test if lateral line defects are responsible for the hyper-inflation phenotype seen in lhfpl5b mutants, we ablated lateral line hair cells in wild type larvae using either neomycin sulfate (neo) or copper sulfate (CuSO4) Figure 2B).
Since the anterior lateral line is likely to be the only part of the sensory organ to interact with the air-water interface during surfacing, we predicted that disruption of the head neuromasts will result in hyperinflation and that trunk neuromasts will not play a role (Figure 4, Supplemental Taken together, we concluded that the anterior region of the lateral line system is critical to surface detection during initial swim bladder inflation.

Decreasing surface tension results in over-filling of the swim bladder
Surface tension is a characteristic that makes the air-water interface distinct from the rest of the water column. We predicted that larval fish sense this stimulus with their anterior lateral line neuromasts and that by decreasing the interfacial tension, we would observe abnormal swim bladder inflation in wild type larvae while lateral line mutants would be unaffected. The interfacial tension between oil and water is approximately half that of air and water (Johansen, 1924). We layered oil onto the surface of the embryo media and allowed larvae to perform surfacing behaviors between 2-6 dpf ( Figure 5). Using this experimental design, we found that the average swim bladder volume of wild types exposed to an oil-water interface (M = 0.016 mm 3 , SD = 0.0029 mm 3 , n = 32) was significantly greater than the control group with access to an air-water interface (M = 0.007 mm 3 , SD = 0.001 mm 3 , n = 28) (One-way ANOVA with Tukey post-test, p < 0.0001).
Furthermore, oil-exposed wild types had statistically similar swim bladder volumes to that of lhfpl5b mutants (M = 0.014 mm 3 , SD = 0.0038 mm 3 , n = 24) (One-way ANOVA with Tukey posttest, p = 0.886) ( Figure 5C). Lateral line mutants overfilled their swim bladders to statistically similar proportions when presented with either an air-water or oil-water interface (M = 0.01 mm 3 , SD = 0.0026 mm 3 , n = 28) (One-way ANOVA with Tukey post-test, p = 0.133). We concluded that surface tension is one of the surface characteristics the lateral line is capable of detecting during initial swim bladder inflation.  Figure 4B). CuSO4 treatments in lhfpl5b mutants did not kill hair cells due to the non-functional MET channel, but rather acted as a control for potential behavioral side effects of the CuSO4 treatments. Overall, optogenetic activation of mechanically-insensitive lateral line organs in lhfpl5b mutants prevented the hyperinflation phenotype. Together, these data support the hypothesis that the lateral line provides crucial sensory information during initial swim bladder inflation in larval zebrafish.

Lateral line mutants exhibit abnormal surfacing behaviors
Our results suggested that larval fish are using their head neuromasts to sense the interfacial tension between air and water at the surface. Even in wild type zebrafish, the interaction with the surface during initial swim bladder inflation is not well characterized. To study this behavior in more detail, we quantified the number of unique visits to the surface and the amount of time per visit spent within the meniscus region from videos of wild type (n = 7) and mutant (n = 14) larvae at 4 dpf. These counts excluded larvae (n = 5 total: 1 wild type and 4 mutant) that under-inflated due to lack of surfacing events. lhfpl5b mutants that over-inflate their swim bladders made more  Figure 7B). Since excess surfacing could be due to some mutants initiating the behavior earlier, we also documented the time of the first surfacing event and found no significant difference between neutrally buoyant and over-inflated larvae ( Figure 7C). We concluded there is a correlation between how larvae interact with the surface during the initial swim bladder inflation period and their resulting swim bladder volume and buoyancy status.

Discussion
The intake of exogenous air from the surface is a common feature for intitial swim bladder inflation in many species of fish (Furukawa et al., 2022;Goolish and Okutake, 1999;Jones and Marshall, 1953;Korsøen et al., 2012;v. Ledebur and Wunder, 1937). However, few studies have considered what sensory information is required to achieve neutral buoyancy. In this study, we describe a new role for the mechanosensory lateral line in mediating initial inflation of the swim bladder in zebrafish (Figure 8). Our data support a model in which larval zebrafish use their head neuromasts to detect the air-water interface in support of their transition to neutral buoyancy.
Lateral line defects in lhfpl5b mutants are responsible for hyperinflation phenotypes lhfpl5b mutant zebrafish are the first genetic model for the specific loss of lateral line function in any organism (Erickson et al., 2020). These "lateral line mutants" exhibit an uncommon swim bladder hyperinflation phenotype that prompted us to investigate this novel connection between lateral line sensation and initial inflation of the swim bladder (Figure 1). Previous studies have demonstrated that restricting access to surface air during the initial inflation period prevents swim bladder inflation in larval zebrafish (Goolish and Okutake, 1999;Lindsey et al., 2011). In addition, one study showed that some solute carrier family 12 member 2 (slc12a2 / nkcc1) mutant zebrafish over-fill their swim bladders and that blocking access to the surface prevents this phenotype (Abbas and Whitfield, 2009). This gene is implicated in endolymph production in the inner ear, but further studies are required to determine the cause of over-inflation in slc12a2 mutants. Likewise, we find that blocking access to the air-water interface eliminates over-inflation in lateral line mutants and greatly decreases average swim bladder volumes of both mutant and wild type larvae (Figure 2). Our method of blocking access to the air-water interface resulted in a small proportion of larvae (both wild type and mutant) inflating their swim bladder to normal proportions, though hyperinflation was never observed. Inflation was likely achieved by larvae accessing air bubbles that remain in the water after submerging the filter (Goolish and Okutake, 1999). Overall, the hyperinflation phenotype in lateral line mutants requires an intake of exogenous air, pointing to a sensory defect in lhfpl5b mutants rather than a physiological one.
In support of this sensory-deficit hypothesis, we show the hyperinflation phenotype is prevented by either transgenic rescue of lhfpl5 function specifically in hair cells or optogenetic stimulation of mechanically-insensitive lateral line hair cells in lhfpl5b mutants (Figures 3 and 6).
Furthermore, chemical ablation of lateral line hair cells in wild type larvae prior to initial inflation can phenocopy the lhfpl5b mutant. A single treatment with CuSO4 caused similar swim bladder over-inflation proportions to lateral line mutants (Supplemental Figure 2). Repeated neomycin treatments over 3-4 dpf caused the next highest percentage of swim bladder over-inflation and a single neomycin treatment only resulted in a few cases of wild type over-inflation. These results agree with previous work showing rapid hair cell regeneration following neomycin treatment and comparatively slower recovery (approximately 24 hours for full recovery) after CuSO4 exposure (Hernández et al., 2006;Mackenzie and Raible, 2012;Murakami et al., 2003;Santos et al., 2006;Venuto and Erickson, 2021). Thus, via multiple genetic and chemical approaches, our data show that lateral line hair cell activity can modulate swim bladder inflation in larval zebrafish.
Ototoxins have been extensively used to study lateral line-mediated behaviors in freeswimming larvae and adult fish (Baker and Montgomery, 1999;Bleckmann, 2006;Coffin and Ramcharitar, 2016;Montgomery et al., 1997;Pavlov and Tyuryukov, 1993). However, we are not aware of any behavioral studies where the lateral line was disrupted prior to larvae achieving neutral buoyancy, likely explaining why this connection between the lateral line and initial swim bladder inflation has not been noted in current literature. Interestingly, an 1896 study predicted a relationship between the lateral line and swim bladder inflation, since disruption of the lateral line in adult goldfish resulted in "swelling of the swim bladder" and positive buoyancy (Richard, 1896).
However, it is not clear if the phenotypic similarities between these adult goldfish and our experimental larval zebrafish share the same underlying causes. Regarding roles for the lateral line in early life behaviors, a close parallel to our findings is the report that unhatched red-eyed treefrogs use their lateral line organ to mediate mechanosensory-cued hatching (Jung et al., 2020). In this case, the frog lateral line can sense vibrational stimuli, such as that produced by predators, and promote escape hatching behaviors even before the onset of vestibular function. Similarly, our data suggest that the lateral line is modulating early locomotor activities in zebrafish prior to the development of fully formed vestibular or rheotactic responses (Bagnall and Schoppik, 2018;Mo et al., 2010;Olive et al., 2016). As such, the involvement of lateral line in the surfacing behavior may represent the earliest behavioral role for the lateral line yet described.

Head neuromasts are required for normal swim bladder inflation, likely by sensing surface tension
We expanded on the full-body lateral line ablation experiments with specific ablations of either the head or truck neuromasts with CuSO4. Previous studies have shown that head neuromasts are involved with detection of surface features and prey capture (Bleckmann, 1988;Carrillo and McHenry, 2016;Müller and Schwartz, 1982;Schwartz and Hasler, 1966). Indeed, ablation of the head neuromasts in wild type larvae yielded similar swim bladder hyperinflation levels to larvae that experienced full body treatments. While this specific experiment did not include lhfpl5b mutants, we find that head-treated larvae generally recapitulated the hyperinflation phenotype of lhfpl5b mutants. Ablation of the tail neuromasts had no effect on either swim bladder volume or buoyancy (Figure 4). These findings suggest that the head neuromasts interact with the air-water interface during surfacing and that the posterior lateral line is dispensible for achieving neutral buoyancy. As such, our findings also provide additional support for the anterior lateral line as a central mediator of surface-related behaviors in fish.
The interfacial tension between air and water represents a unique hydrodynamic stimulus that may be detected by the lateral line. The tension between oil and water is half that of air and water (Johansen, 1924). Thus, by adding a layer of oil to the surface, we predicted that surface detection would become increasingly difficult and result in over-filling of the swim bladder. We find that, when given access to an oil-water interface, wild type larvae over-fill their swim bladders with oil to a statistically similar volume as lateral line mutants who overinflate with air ( Figure 6).
We interpret this result as support for the hypothesis that the lateral line senses interfacial tension between air and water as larvae interact with the surface during initial swim bladder inflation. This interpretation is further supported by the observation that lhfpl5b mutants (who are already insensitive to interfacial tension) exhibit similar swim bladder volumes when exposed to either oil or air. However, a possible complementary interpretation for these data is that larvae who fill their swim bladders with oil may be deprived of the normal feedback on buoyancy levels as they intake from the surface. Since oil is denser than air, oil-filled larvae are negatively buoyant. In this scenario, larvae may continue to visit the surface for air intake in an attempt to achieve neutral buoyancy. However, future studies are required to examine if such a feedback mechanism exists in larval zebrafish.

Swim bladder over-inflation is correlated with excess time spent at the air-water interface
Since our data suggest that the air-water interface is providing a distinct stimulus to the lateral line, we investigated whether surface interactions differed between hyperinflated lhfpl5b mutants and their wild type counterparts. As predicted, over-inflated mutants took more visits and spent more time at the surface than neutrally-buoyant mutant and wild type siblings (Figure 7).
Excess breaching of the surface occurred despite over-inflated and neutrally buoyant larvae exhibiting similar timing for their first surfacing attempts. While it is unclear whether larvae ingest air during the entirety of the duration spent above the air-water interface, it is known that surface breaches are observed during initial inflation (Goolish and Okutake, 1999;Lindsey et al., 2010;Rieger and Summerfelt, 1998). Therefore, the increase in surface visits likely leads to an increase in air deposition to the swim bladder in lateral line mutants.

Multisensory integration during the surfacing behavior
Interestingly, approximately half of all lateral line mutants inflate their swim bladders to normal proportions and continue to develop into viable adults. To account for this fact, we hypothesize that additional somatosensory, photosensory, or mechanosensory systems may compensate for the loss of lateral line sensation and help mutant larvae achieve neutral buoyancy (Riley and Moorman, 2000;Suchocki and Sepulveda-Villet, 2019;Trotter et al., 2003). The lateral line is involved several multi-sensory behaviors, including flow orientation (Bak-Coleman et al., 2013;Coombs et al., 2020;Liao, 2006), prey capture (Carrillo andMcHenry, 2016;Nelson et al., 2002;New, 2002), and predator avoidance (Free et al., 2019;Jung et al., 2020;Stewart et al., 2013), and we predict that surfacing can be added to this list.
The photosensory and mechanosensory systems exhibit distinct organizational patterns for primary input to the central nervous system -even the anterior and posterior lateral line ganglia primary projections are somatotopically organized in zebrafish larvae (Alexandre et al., 1999;Fame et al., 2006;Hale et al., 2016;Liao and Haehnel, 2012;McCormick, 1989;Mirjany and Faber, 2011). The Mauthner neurons integrate multisensory input at the level of the hindbrain, but the surfacing behavior is unlikely to use this circuit. For higher order processing however, information from the lateral line, vestibular, and photosensory systems are integrated in various brain regions. The thalamus is one such sensory integration center in zebrafish (Mueller, 2012).
Socially-relevant mechanosensory and photosensory information is represented in a thalamic region marked by the expression of parathyroid hormone 2 (pth2) and somatostatin 7 (sst7) (Anneser et al., 2020;Kappel et al., 2022;Venuto et al., 2022). Vestibular information is also represented in the thalamus (Favre-Bulle et al., 2018;Migault et al., 2018), though it is not clear if the exact same region is involved. Photosensory, lateral line, and vestibular/auditory information is also integrated in the tectum (Favre-Bulle et al., 2018;Filosa et al., 2016;Isa et al., 2021;Migault et al., 2018). In fact, neurons in the periventricular layer (PVL) of the tectum, which is similar to the superior colliculus in mammals, respond to visual, auditory, and lateral line inputs (Ingle, 1973;King et al., 1996;Perrault et al., 2011;Thompson et al., 2016). However, it is hypothesized that most of these modalities are processed in parallel at younger stages of development (Thompson et al., 2016). It is worth noting that these sensory systems are in developing stages during surfacing behaviors.
Photosensory cues did not alter the buoyancy status of either wild type or lhfpl5b mutant larvae (Supplemental Figure 1), suggesting that, at most, photosensory systems play a subtle modulatory role in the surfacing behavior of larval zebrafish. The vestibular system is likely required to coordinate the vertical orientation and climbing portions of surfacing (Bagnall and Schoppik, 2018;Ehrlich and Schoppik, 2017;Ehrlich and Schoppik, 2019). Meanwhile, our data support the idea that the lateral line is responsible for the surface detection and breaching aspects of surfacing. However, given that both the vestibular and lateral line systems are providing spatial information in during the surfacing behavior, there may be crosstalk between them. For example, the vestibular system must quickly adapt to a dynamic increase in buoyancy over the course of multiple breaching events, so feedback from the lateral line during surface interactions may help fine tune the vestibular sense. However, the link between these systems have not been wellinvestigated during deliberate behaviors like surfacing (Chagnaud et al., 2017;Liu et al., 2021).
So, while sensory integration exists in zebrafish and seems necessary for larvae to achieve neutral buoyancy, the integration dynamics are unclear during the intial swim bladder inflation timeframe and require future investigation. Line of best fit applied through the combination of mutant and wild type data (linear model, Rsquared = 0.8574). C) Percentage of larvae that exhibit over-, regular, and under-inflation of the swim bladder, as defined by positive, neutral, and negative buoyancy. Legend from panel D applies to both panel C and D. A Chi-Squared Test was used to determine significance. D) Swim bladder volume (mm 3 ) in wild type and lhfpl5b mutant larvae at 6 dpf. A Welch's t-test was used to determine significance. *** = p < 0.001, **** = p < 0.0001. Scale bar = 0.2 mm.

Figure 2:
Blocking access to the surface eliminates the hyperinflation phenotype in lateral line mutants (lhfpl5b -/-). A) Diagram of experimental set up with a filter blocking access to the surface. B) Percentage of larvae that exhibit over-, regular, and under-inflation of the swim bladder at 6 dpf. A Chi-Squared Test was used to determine significance. C) Swim bladder volume (mm 3 ) of larvae at 6 dpf. A One-Way ANOVA was used to determine significance. **** = p < 0.0001, ** = p < 0.01, ns = not significant. Full ANOVA table in supplemental tables.

Figure 3:
Rescue of lateral line mutants (lhfpl5b -/-) with the Tg(myo6b:EGFP-lhfpl5a)vo23 transgene. A) Percentage of larvae that exhibit swim bladder under, regular, and over-inflation at 6 dpf. A Chi-Squared Test was used to determine significance. B) Swim bladder volume (mm 3 ) of larvae at 6 dpf. A One-Way ANOVA was used to determine significance. **** = p < 0.0001, ns = not significant. Full ANOVA table in supplemental tables.

Figure 4:
Head-specific ablations of the lateral line recapitulates the lhfpl5b mutant hyperinflation phenotype. A) Diagram of experiment. B) Percentage of larvae that exhibit swim bladder under, regular, and over-inflation at 5 dpf. A Chi-Squared Test was used to determine significance. C) Swim bladder volume (mm 3 ) of larvae at 5 dpf. A One-Way ANOVA was used to determine significance. * = p < 0.05, , **** = p < 0.0001, ns = not significant. Full ANOVA table in supplemental tables. Images of live larvae following treatment in Supplemental Figure 3.

Figure 5:
Decreasing interfacial surface tension with mineral oil results in over-filling of the swim bladder. A) Diagram of experiment with 2 mm layer of oil on surface. B) Images of swim bladder phenotypes resulting from surface oil and surface air exposure in wild type larvae at 6 dpf. C) Swim bladder volume (mm 3 ) at 6 dpf. A One-Way ANOVA was used to determine significance. * = p < 0.05, ns = not significant. Scale bar = 0.1 mm. Full ANOVA table in supplemental tables.

Figure 6:
Optogenetic activation of lateral line hair cells with Channelrhodopsin-2 (ChR2) in wild type and lhfpl5b mutant larval zebrafish. A) Diagram of experiment with blue light stimuli delivered for 25 milliseconds at 1 second intervals. Treatments were started at 4 dpf and swim bladder volume was assessed at 6 dpf. B) Swim bladder volume (mm 3 ) of larvae at 6 dpf, n = 16-21 per condition. One-Way ANOVA was used to determine significance. ** = p < 0.001, ns = not significant. Full ANOVA table in supplemental tables.

Figure 7:
Quantification of the surfacing behaviors of wild type and lhfpl5b mutant zebrafish larvae during initial swim bladder inflation. A) Total number of individual visits taken by larval fish to the air-water interface on 4 dpf for 12 hours (9 AM -9 PM). B) Total amount of time larval fish spend at the surface for all visits combined on 4 dpf. C) Time to first surface visit after access was allowed. A One-Way ANOVA was used to determine significance. *** = p < 0.001, ns = not significant. Supplemental Figure 2. Ototoxic ablations of the lateral line. A) Percentage of larvae that exhibit swim bladder under, regular, and over-inflation. A Chi-Squared Test was used to assess significance. B) Swim bladder volume (mm 3 ) in 5 dpf larvae. A One-Way ANOVA was used to determine significance. **** = p < 0.0001, *** = p < 0.001, ** = p < 0.01, ns = no significance. A full ANOVA table can be found in the supplemental tables.
Supplemental Figure 4: Channelrhodopsin-2 (ChR2) expression in Tg(lhfpl5b2028:ChR2-EYFP-pA) transgenics. A) Images of ChR2-YFP in hair cells in a 2 dpf neuromast, utricle, and crista where fluorescence expression is only seen in the neuromasts of the lateral line. First panel is YFP only (false-colored green) and the second panel is a merge of YFP and DIC. Scale bars = 10 uM. B) Swim bladder volume (mm 3 ) of 6 dpf larvae from the experimental condition (blue light overhead). A One-Way ANOVA was used to determine significance. ** = p < 0.00724, ns = no significance. A full ANOVA table can be found in the supplemental tables. C) Percentages and box plots of experiment in Figure 6 to show inflation categories. D) Percentages and box plots of experiment in Supplemental Figure 4B to show inflation categories.