Use of the swim bladder and lateral line in near-field sound source localization by fish

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1 2014. Published by The Company of Biologists Ltd (2014) 217, doi: /jeb RESEARCH ARTICLE Use of the swim bladde and lateal line in nea-field sound souce localization by fish Allison B. Coffin 1,2, *,, David G. Zeddies 3, Richad R. Fay 4, Andew D. Bown 5,, Pete W. Aldeks 6, Ashwin A. Bhandiwad 6, Robet A. Moh 6, Michael D. Gay 7, Pete H. Roges 7 and Joseph A. Sisneos 1,6,8, ABSTRACT We investigated the oles of the swim bladde and the lateal line system in sound localization behavio by the plainfin midshipman fish (Poichthys notatus). Repoductive female midshipman undewent eithe sugical deflation of the swim bladde o cyoablation of the lateal line and wee then tested in a monopola sound souce localization task. Fish with nominally deflated swim bladdes pefomed simila to sham-deflated contols; howeve, postexpeiment evaluation of swim bladde deflation evealed that a majoity of deflated fish (88%, seven of the eight fish) that exhibited positive phonotaxis had patially inflated swim bladdes. In total, 95% (21/22) of fish that localized the souce had at least patially inflated swim bladdes, indicating that pessue eception is likely equied fo sound souce localization. In lateal line expeiments, no diffeence was obseved in the popotion of females exhibiting positive phonotaxis with ablated (37%) vesus sham-ablated (47%) lateal line systems. These data suggest that the lateal line system is likely not equied fo sound souce localization, although this system may be impotant fo fine-tuning the appoach to the sound souce. We found that midshipman can solve the 180 deg ambiguity of souce diection in the shallow wate of ou test tank, which is simila to thei nesting envionment. We also found that the potential diectional cues (phase elationship between pessue and paticle motion) in shallow wate diffes fom a theoetical fee-field. Theefoe, the geneal question of how fish use acoustic pessue cues to solve the 180 deg ambiguity of souce diection fom the paticle motion vecto emains unesolved. KEY WORDS: Heaing, Lateal line, Phonotaxis, Swim bladde INTRODUCTION Fish ae well equipped to detect undewate acoustic and vibatoy stimuli. Most teleost fishes have both auditoy and lateal line systems, and each system is esponsive to the local fields poduced by acoustic and/o vibatoy souces. These systems ae used in a 1 Viginia Meill Bloedel Heaing Reseach Cente, Univesity of Washington, Seattle, WA 98195, USA. 2 Depatment of Otolayngology Head and Neck Sugey, Univesity of Washington, Seattle, WA 98105, USA. 3 JASCO Applied Sciences, Silve Sping, MD 20902, USA. 4 Maine Biological Laboatoy, Woods Hole, MA 02543, USA. 5 Depatment of Speech and Heaing Sciences, Univesity of Washington, Seattle, WA 98105, USA. 6 Depatment of Psychology, Univesity of Washington, Seattle, WA 98195, USA. 7 Geoge W. Wooduff School of Mechanical Engineeing, Geogia Institute of Technology, Atlanta, GA 30332, USA. 8 Depatment of Biology, Univesity of Washington, Seattle, WA 98195, USA. *Pesent addess: Washington State Univesity Vancouve, Vancouve, WA 98686, USA. Pesent addess: Univesity of Coloado School of Medicine, Auoa, CO 80045, USA. These authos contibuted equally to this wok Autho fo coespondence (sisneos@u.washington.edu) Received 11 July 2013; Accepted 13 Mach 2014 vaiety of behavioal contexts, including behavios such as pey detection and mate selection, which necessitate sound souce oientation and localization (Fay, 2005; Webb et al., 2008). While seveal studies have established that some fishes can detemine sound diection and localize undewate sound souces (Schuijf, 1975; Schuijf and Hawkins, 1983; Zeddies et al., 2010; Zeddies et al., 2012), thee is much debate ove the mechanism(s) undelying sound souce localization ability (see Fay and Simmons, 1999; Roges and Zeddies, 2008). Sound pessue is a scala quantity containing no infomation about diection, and thus is in itself not useful fo detemining sound souce diection. The paticle motion component of sound, in contast, is a vecto quantity (containing a diectional component) and could thus be useful fo detemining the diection to a sound souce. Fishes possess two systems fo paticle motion detection, the inne ea and the lateal line (Baun and Coombs, 2000; Baun and Coombs, 2010). Based on his wok on the lateal line ogans of killifish (Fundulus heteoclitus), van Begeijk suggested that pessue eception by the inne ea allowed fo detection of a sound souce but that the lateal line system was esponsible fo supplying diectional infomation (Hais and Van Begeijk, 1962; Van Begeijk, 1967). Yeas late, the elative contibutions of the inne ea and lateal line to paticle motion detection and souce localization ae still not fimly undestood. Paticle motion detection by the inne ea alone seems insufficient fo signaling the diection to a sound souce because the axis of the paticle motion vecto points both towads and away fom the souce, meaning thee is a 180 deg ambiguity that emains unesolved without futhe infomation (Fay, 2005). In othe wods, fish sensitive only to paticle motion should be able to identify the axis along which the local paticle motion lies, but unable to distinguish whethe the souce is located at one heading o at 180 deg in the opposite diection. Cuent models fo sound souce localization by fish depend on the detection and pocessing of both the pessue and paticle motion components of sound fo the esolution of this ambiguity (Fay, 2005). A majo assumption of seveal elated hypotheses (Chapman and Hawkins, 1973; Schuijf, 1975), including a dominant hypothesis of sound souce localization known as the phase model (Schuijf and Buwalda, 1975), holds that fishes ae able to use the phase diffeence of sound pessue and paticle motion components to compute the diection to a sound souce (esolving the 180 deg ambiguity). Howeve, these models assume a fa-field pessue paticle velocity elationship and equie sinusoidal stimuli. An altenative computational model poposed by Roges et al. (Roges et al., 1988) fo esolving the 180 deg ambiguity also equies that fish detect both sound pessue and paticle motion, but woks at all distances fom a simple monopole sound souce and does not equie sinusoidal signals. Fo sinusoidal signals, the Schuijf and Roges models make identical pedictions, dependent on the phase diffeence of the pessue and paticle velocity components. 2078

2 (2014) doi: /jeb It is highly likely that all fishes ae able to detect the paticle motion component of sound via the inne ea otolithic end ogans, which function as inetial acceleometes (De Vies, 1950; Dijkgaaf, 1960; Fay, 1984). Recent behavioal studies with the plainfin midshipman fish (Poichthys notatus Giad 1854) show that these fish can use local acoustic paticle motion to locate a sound souce (Zeddies et al., 2010; Zeddies et al., 2012). The extent to which midshipman fish ae eceptive to sound pessue, o able to esolve the 180 deg ambiguity poblem via pessue eception, is not known. Moe geneally, the ole that pessue eception o the mechanosensoy lateal line, which is sensitive to both paticle motion and local pessue gadients (Webb et al., 2008), may play in sound souce localization emains empiically untested. Hee, we investigate the use of the swim bladde and lateal line in sound souce localization by female plainfin midshipman in the nea field, wheein the souce distance is less than an acoustic wavelength and the atio of pessue to adial paticle velocity is a distance-dependent complex value smalle in magnitude than the density sound speed poduct. Expeiments wee conducted duing the summe of 2010 on wild-caught females in epoductive condition. Acoss expeiments, stimuli wee simulated advetisement calls of male midshipman fish, which ae appoximately sinusoidal (Bass, 1992), and thus well suited to test the pedictions of sound souce localization models. Ou findings suggest that (1) pessue eception is likely equied fo nea-field souce localization, (2) midshipman can esolve the 180 deg ambiguity, though the mechanisms fo doing so emain obscue, and (3) the lateal line system may not be necessay fo nea-field souce localization. RESULTS Acoustic field chaacteization The spatial chaacteistics of the acoustic field geneated by the J-9 and AQ339 sound pojectos wee quantified fom the tank mapping measuements made with miniatue hydophones (sound pessue) and a ti-axial acceleomete (paticle motion). Fo acs of constant adius fom the souce, the azimuthal standad deviation of the pessue at 90 Hz was found to be no moe than 10% and 6% fo the J-9 and AQ339 pojectos, espectively. Standad deviation values at 75 and 80 Hz fo the AQ339 wee 9% and 6%, espectively. These esults confimed that the sound fields wee cicumfeentially unifom (axisymmetic) within the mapped aea of the sound field that contained the fish elease points and phonotaxic paths of the tested fish. Biased and unbiased elease expeiments Sound playback expeiments wee conducted at night in an outdoo cylindical concete tank, with initial elease sites and the speake position as indicated in Fig. 1. In this fist expeiment, we asked whethe thee was a diffeence in localization behavio fo fish that wee eleased so that thei initial swimming diection was biased towads the sound souce (biased elease) vesus fish that wee allowed to leave the elease site in any diection (unbiased elease). The phonotaxic esponses displayed in the biased vesus unbiased elease expeiments wee unambiguous; positive phonotaxic esponses entailed epeated contact with the souce by the fish [see Zeddies et al. (Zeddies et al., 2010) fo desciptions of the phonotaxic esponse]. Of the 17 epoductive females tested in the unbiased (open) elease, 65% (n=11) exhibited a positive phonotaxic esponse. Of the 31 epoductive females tested in the biased elease expeiments, 61% (n=19) exhibited positive phonotaxic esponses. A logistic egession showed that thee was Sceen A Speake 44 cm 44 cm 74.5 cm Fig. 1. Schematic of the expeimental setup (tank diamete=4 m, wate depth=50 cm). Shown is the monopole sound souce (Lubell AQ339, Clak Synthesis, Littleton, CO, USA, o a US Navy J-9 tansduce), the opaque sceen and the animal elease sites (A and B). no diffeence in the popotion of females that exhibited positive phonotaxic esponses in the biased (61%) elease expeiments compaed with the unbiased (65%) elease expeiments [β=0.14±0.67 (mean ± s.e.m.), t=0.23, P=0.82], suggesting that fish wee able to esolve the 180 deg ambiguity. Swim bladde deflation expeiments Phonotaxic esponses wee next examined in fish with intact o deflated swim bladdes to detemine whethe the swim bladde was equied fo nea-field sound souce localization. Of the 28 epoductive females with sham-deflated swim bladdes, 50% (n=14) exhibited positive phonotaxic esponses; the tajectoies these females took afte elease fom Site B ae shown in Fig. 2A. Of the 21 epoductive females that undewent swim bladde deflation sugey, 38% (n=8) exhibited positive phonotaxic esponses (see Fig. 2B). An initial logistic egession analysis indicated no diffeence in the popotion of females with deflated (38%) and sham-deflated (50%) swim bladdes that exhibited positive phonotaxis [β=0.49±0.59 (mean ± s.e.m.), t=0.83, P=0.41]. Howeve, upon postmotem examination of the fish that undewent swim bladde deflation sugey, we found that 12 (out of 21) had patially (25 to 50%) to nealy completely (>90%) inflated swim bladdes. The obsevation of patial swim bladde inflation within 24 h post deflation sugey (and <12 h post-test) suggests apid einflation of the swim bladde in this subset of animals. We thus compaed, post hoc, fish with e-inflated swim bladdes (fish with geate than 25% swim bladde e-inflation, n=12) against those with still-deflated swim bladdes (fish with 100% swim bladde deflation). Wheeas 58% (n=7) of e-inflated fish exhibited positive phonotaxis a popotion not significantly diffeent fom that in sham-deflated contols (β= 0.34±0.67, t=0.48, P=0.68) only one of nine still-deflated fish exhibited positive phonotaxis a popotion significantly lowe than in the e-inflated sub-goup (β=2.41±1.21, t=1.99, P<0.05). In sum, we found that 95% (21/22) of fish that localized the souce had at least patially inflated swim B 2079

3 (2014) doi: /jeb Distance (cm) A Distance (cm) B Negative esponse (deflated SB) Positive esponse (deflated SB) Negative esponse (einflated SB) Positive esponse (einflated SB) Fig. 2. Phonotaxic esponse pathways of epoductive female midshipman fish that undewent swim bladde deflation sugey. (A) Response pathways of females with sham-deflated swim bladdes that exhibited positive (geen cicles) phonotaxis and negative (ed cicles) esponses to the simulated advetisement call stimulus. (B) Response pathways of females with deflated and e-inflated swim bladdes (see Results) that exhibited positive phonotaxis with eithe deflated (geen cicles) o e-inflated (blue tiangles) swim bladdes and negative esponses with eithe deflated (ed squaes) o e-inflated (oange diamonds) swim bladdes. The axes ae the distance fom the cente of the tank (cm), whee the monopole J-9 speake (black squae) was located, and the dotted line epesents the position of the opaque sceen. Gey cicles indicate the position of the plastic mesh cylinde whee the animals wee eleased. bladdes, indicating that pessue eception is likely equied fo sound souce localization. Lateal line ablation expeiments Mophology Phonotaxis esponses wee next examined in fish with ablated lateal line systems in ode to detemine the elative contibution of the lateal line to souce localization. Lateal line ablation was achieved with a liquid N 2 -dipped pobe. Cyoablation success was qualitatively veified with DASPEI in a subset of fish immediately afte sound souce localization expeiments wee pefomed (see Fig. 3 fo an illustation of lateal line mophology). DASPEIlabeled neuomasts wee pesent in steeotyped pattens on the head, tunk and tail of intact and sham-ablated fish, while DASPEI labeling was lagely absent fom neuomasts in ablated fish (Fig. 4). Only supeficial neuomasts (SN) could be veified in this way because of the dak skin pigmentation of the skin that obscued canal neuomast (CN) visualization, and because of the supeglue ove the canals in ablated animals. SN ablation was quantified in fixed FM 1-43FX-labeled animals as descibed in the Mateials and methods (Fig. 5, Table 1). Regions used fo quantification ae indicated in Fig. 3. Thee wee 16.1±6.7% SN (mean ± s.d.) emaining in liquid N 2 -ablated, FM-labeled fish (n=9 fish, SN quantified pe fish), with the highest pecentage of intact neuomasts obseved on the mandible (38.9±15.8%) and the lowest pecentage seen on the dosal tunk line (4.9±6.3%; Table 1). Two SN 4 SN 1 CN CN SN 2 SN 3 SN 4 Fig. 3. Schematic illustation of the plainfin midshipman fish lateal line mechanosensoy system (adapted fom Geene, 1899). All illustated neuomasts and seveal stitches not illustated [supeficial neuomasts (SN) pimaily on the head and undebelly] wee tageted fo ablation. Fou egions of SN wee selected fo phalloidin labeling (neuomasts filled in black): a 2 cm egion fom the ight dosal line stating at the anteio edge of the dosal fin (SN 1), a 1 cm egion on the top of the head (left side; SN 2), cm fom the vental line (left side) stating at the anteio edge of the anal fin (SN 3), and the entie caudal fin (SN 4). Given the small numbe of canal neuomasts (CN) in this species, all canals wee dissected fom the head and labeled athe than selecting discete CN egions fo sub-sampling (aows; neuomasts within canals not illustated). Fig. 4. DASPEI labeling of neuomasts in live, anesthetized female midshipman. Images ae fom the dosal lateal line that uns along the base of the dosal fin. (A) Intact animal; (B) sham-opeated animal (vental photophoes ablated); (C) lateal line ablation pefomed same day; (D) lateal line ablation pefomed 1 day pio to behavioal testing and imaging. Aows in C and D delineate examples of neuomast locations. Aowheads in B and D indicate photophoes, which ae autofluoescent. Scale ba in A, 500 μm (applies to all panels). 2080

4 (2014) doi: /jeb mophology suounding the usual hai-cell-containing egion (Fig. 6F). Intact CN wee occasionally detected in ablated fish at a fequency of ~1 intact CN pe animal; howeve, CN wee not quantified as we could not be cetain that some wee not damaged duing postmotem dissection. It is unlikely that the few emaining CN in ablated fish eceived any stimulation because of the supeglue that obstucted the incuent and excuent openings to the canals. Fig. 5. FM 1-43FX-labeled neuomasts in fixed animals. Images ae fom the ventomedial suface between the pectoal fins. (A) Intact animal; (B) sham-opeated animal; (C,D) lateal line ablation pefomed same day; (E) lateal line ablation pefomed 1 day pio to behavioal testing and imaging. White aows in C E show examples of neuomast locations whee all hai cells wee successfully ablated. Black aows in D point to emaining neuomasts in a fish subjected to liquid N 2 ablation. Scale bas, 500 μm. fish with 17.4% and 9.6% intact SN failed to localize the sound souce, while a fish with only 6.8% emaining SN localized the sound souce, suggesting that the positive phonotaxis seen in many of the ablated fish was not due to excess intact neuomasts. Neuomast stuctue was visualized in both SN and CN using phalloidin labeling, as shown in Fig. 6. The small papillae ae clealy visible in intact SN, with a patch of hai bundles lying between the two papillae (Fig. 6A,B). Intact CN ae lage and have many moe hai bundles than SN (compae Fig. 6B and 6C) and lack peipheal papillae. These papillae ae damaged in ablated SN and the hai bundles and suounding epithelial laye ae absent (Fig. 6D,E). Ablated CN can be ecognized by the epithelial Localization behavio Of the 35 epoductive females that had thei lateal line system ablated, 37% (n=13) exhibited positive phonotaxic esponses. The pathways these females took afte elease fom Site A ae shown in Fig. 7A. Of the 15 epoductive females that had sham-ablated lateal lines (photophoes only), 47% (n=7) exhibited positive phonotaxic esponses. Swimming pathways fo these fish afte elease fom Site A ae shown in Fig. 7B. Because some expeiments wee conducted the day afte lateal line ablation, a hieachical logistic egession was pefomed to contol fo the effect of day. Thee was a significant incease in esponse on the day afte lateal line ablation in both lateal-line-ablated (28% on Day 1, 55% on Day 2) and sham-ablated animals (28% on Day 1, 50% on Day 2) [β=1.75±0.75 (mean ± s.e.m.), t=2.33, P=0.02]. Howeve, contolling fo the effect of day, thee was no diffeence in the popotion of females with ablated (37%) and sham-ablated (47%) lateal lines that exhibited positive phonotaxis (β= , t=0.97, P=0.33). A mean vecto analysis of the swimming pathways at the initial elease showed that the diection of movement was biased towads the sound souce (P<0.001 fo both ablated and shamablated lateal line females). The angles that the positive phonotaxic fish took (afte eleased fom Site A) wee compaed fo females with ablated and shamablated lateal line systems. The mean oientation eo elative to the souce was significantly diffeent at the initial elease fo latealline-ablated females (mean oientation eo=39.0 deg) compaed with lateal line sham-ablated females (mean oientation eo=15.3 deg) (Watson Williams test, F 1,18 =6.3, P<0.025). In addition, the mean oientation elative to the souce at the midpoint of the phonotaxic pathway taken was also significantly diffeent fo lateal-line-ablated females (mean oientation eo=33.4 deg) compaed with lateal line sham-ablated females (mean oientation eo=12.8 deg) (Watson Williams test, F 1,18 =6.39, P<0.025). DISCUSSION The plainfin midshipman fish is a good model to exploe how fishes localize undewate sound souces (Zeddies et al., 2010; Zeddies et al., 2012), in pat because epoductive females exhibit obust phonotaxic esponses to the undewate acoustic playback of natual and synthetic male advetisement calls (Bass et al., 1999; McKibben and Bass, 2001). Peviously, we demonstated that the plainfin midshipman fish can use local acoustic paticle motion to guide the animal to a sound souce (Zeddies et al., 2010; Zeddies et al., 2012), but the contibution of swim bladde pessue cues and the mechanosensoy lateal line to sound souce localization emained unesolved. Thus, the pimay objective of this study was to detemine whethe the swim bladde and/o the lateal line wee equied fo nea-field sound souce localization. Ou findings suggest that pessue eception is likely equied fo nea-field souce localization, that fish can solve the 180 deg ambiguity inheent in the paticle motion vecto, and that the lateal line system is likely not necessay fo nea-field sound souce localization. It is impotant to note, howeve, that these expeiments wee pefomed 2081

5 (2014) doi: /jeb Table 1. Quantification of FM-labelled supeficial neuomasts (SN) fom six egions of liquid N 2 -ablated fish (n=9) Dosal line Top of head Belly Lowe jaw Caudal fin Opeculum Total Fish Intact (%) Total Intact (%) Total Intact (%) Total Intact (%) Total Intact (%) Total Intact (%) Total Intact (%) Total (3.9) 76 0 (0) 13 0 (0) 43 9 (22.5) (33.8) 68 3 (21.4) (15.0) (20.8) 77 0 (0) 9 7 (14) (65.0) (40.0) 25 1 (6.7) (28.3) a 0 (0) 88 0 (0) 10 5 (15.6) (57.1) (29.8) 47 3 (23.1) (17.4) a 0 (0) 73 4 (40) 10 3 (7.1) (33.3) 33 2 (5.3) 38 0 (0) (9.6) (5.2) 77 1 (7.1) 14 0 (0) (52.3) 44 0 (0) 32 1 (5.5) (12.4) b 5 (5.5) 90 0 (0) 13 1 (2.1) (40.0) (31.7) 41 0 (0) (14.2) (4.5) 89 0 (0) 8 0 (0) 50 9 (31.0) (70.0) 40 1 (5.9) (18.0) (2.8) 70 0 (0) (20.0) 50 8 (22.2) (78.6) 42 0 (0) (23.4) (1.6) 62 0 (0) 11 0 (0) (26.3) 38 0 (0) 27 1 (6.7) (6.7) 177 Data ae pesented as the numbe and pecentage of intact SN (as visualized with FM) elative to the total numbe of SN. Neuomast position was detemined by epithelial mophology (e.g. fleshy papillae) and pigmentation diffeences as compaed with the suounding non-sensoy aeas, allowing fo unambiguous identification of SN even in the absence of FM labeling. Data fo sham-ablated animals ae not shown but missing neuomasts wee aely seen in these animals (0 2 unlabeled SN pe fish). a Ablated fish that did not localize the sound souce. b Phonotaxic esponse, FM labeling and euthanasia occued 1 day post-ablation. in a benthic species that inhabits an exteme shallow-wate envionment (duing the beeding season), and theefoe ou esults may not petain to all fishes, especially pelagic species that live in the wate column. Sound pessue eception and esolving the 180 deg ambiguity Duing an acoustic distubance, an infinitesimal paticle of fluid undegoes a small linea displacement of oscillating polaity. In the pesent case, with an axisymmetic sound field, the paticle motion vecto altenately points towads and away fom the acoustic souce fo equal amounts of time. Theefoe, this paticle motion vecto does not indicate which diection along the line to follow in ode to each the souce, giving ise to the so-called 180 deg ambiguity in paticle motion. The behavioal capacity fo diectional-dependent heaing in fish has been demonstated [e.g. diectional masking in Atlantic cod Gadus mohua (Hawkins and Sand, 1977); sound souce localization in plainfin midshipman (Zeddies et al., 2010; Zeddies et al., 2012)], but thee wee no data to demonstate that fish solve the 180 deg ambiguity poblem duing sound souce localization. In the pesent study we found no diffeence in the positive phonotaxic esponse ate when fish wee allowed to swim in any diection upon elease ( unbiased elease) vesus when they wee diected towad the sound souce upon elease ( biased elease). We also found that the positive esponse ate was >60% in both elease cases. If fish could not solve the 180 deg ambiguity, biasing thei elease towad the souce would be expected to incease the positive phonotaxic esponse ate elative to that fo the unbiased elease, as in the unbiased condition, fish able to detect the souce axis but unable to detemine font fom back would be expected to swim away fom the souce in appoximately half of tials. Moeove, no fish wee obseved to exit away fom the sound souce and then coectly tun and move to the sound souce (Fig. 2). Theefoe, because the positive phonotaxic esponse ate was the same in both elease conditions, and the positive esponse ate in the unbiased situation was >60%, we suggest that midshipman effectively esolve the 180 deg ambiguity poblem duing sound souce localization. While it is tue that a subset of fish in all expeiments failed to localize the sound souce, the majoity of these fish did not swim 180 deg in the opposite diection, as would be expected if they wee motivated to locate the souce but could not solve the 180 deg ambiguity. Instead, some of the non-localizing fish emained at the elease site, while the othes swam in vaious diections towads the edges of the tank, at which point the tial was teminated. The elationship between paticle motion and pessue is cental to hypotheses concening the basis of sound souce localization by fish, notably the phase model suggested by Schuijf and Buwalda (Schuijf and Buwalda, 1975) and the moe geneal model of Roges et al. (Roges et al., 1988). In these models, fish must be eceptive to paticle motion and pessue in ode to solve the 180 deg ambiguity. To assess the ole that pessue eception may play in sound souce localization by midshipman, we attempted to deflate the swim bladde in a subset of animals. Collectively, 95% of fish (21/22) that exhibited a positive phonotaxic esponse had at least patially inflated swim bladdes (seven e-inflated, 14 sham deflated), while only a single fish with a fully deflated swim bladde (1/9) exhibited a positive phonotaxic esponse. Taken togethe, these esults suggest that sound pessue is likely an impotant cue used in sound souce localization by these fish. The oveall eduction in phonotaxis and sound localization in fish with intact o e-inflated swim bladdes compaed with females in pevious studies (Zeddies et al., 2010; Zeddies et al., 2012) is likely due to the afte effects of anesthesia and the obseved eduction in swimming behavio in ecently anesthetized midshipman that wee sugically manipulated, a phenomenon also seen in the lateal line ablation expeiments (J.A.S. and A.B.C., data not shown). Although it is appaent that sound pessue is used fo souce localization by the plainfin midshipman fish, it is not clea how the pessue cue is used to solve the 180 deg ambiguity poblem. The phase model (Schuijf and Buwalda, 1975) was developed based on expeiments using appoximately plana wave conditions, wheein sound pessue and adial paticle velocity ae in phase, having the same o opposite polaity depending on the diection of popagation. The popagation diection polaity elationship pesists fo the inphase components of the pessue and paticle velocity even in the nea field fo a fee-field point souce. This is the basis of the Roges et al. (Roges et al., 1988) algoithm fo esolving the ambiguity. Howeve, fo the popagation conditions in ou test tank and fo some natual envionments, including the midshipman nesting envionment, the phase elationship between sound pessue and adial paticle velocity is moe complicated, and can be significantly diffeent fom the field of a fee-field point souce. We discuss these diffeences in depth in a modeling study descibed in the Appendix. In shot, ou esults indicate that if a fish esponded coectly to a fee-field souce (at any ange), it would actually espond in the 2082

6 (2014) doi: /jeb SN Intact Ablated Fig. 6. Phalloidin-labeled neuomasts fom intact (A C) and ablated (D F) animals. (A) Low-magnification image of a vental SN showing intact papillae. The neuomast between these stuctues is indicated with an aow and is shown at highe magnification in B. (C) Intact CN with a full complement of hai bundles. (D) Low-magnification image of a damaged vental SN. The papillae ae still pesent but appea damaged. The aow indicates the position of the ablated neuomast, which is shown in highe magnification in E. (F) Liquid N 2 -ablated CN. The aowhead maks scatteed hai bundle emnants. Scale bas, 100 μm (A,D); 10 μm (B,E); 30 μm (C,F). CN SN wong diection duing phonotaxis in the non-fee-field Bodega Bay tank. Similaly, ou popagation models fo conditions simila to the natual nesting envionment of the midshipman indicate that if a fish esponded in the coect diection fo a fee-field point souce, it would also espond in the wong diection in the nesting envionment at distances that anged fom 1 cm to m Distance (cm) Distance (cm) depending on bottom type. Ultimately, because female midshipman consistently exhibited positive phonotaxis in ou test tank, we ae left to conclude that diffeent souce localization stategies ae used in shallow-wate vesus fee-field envionments, o that midshipman ae unable to coectly esolve the 180 deg ambiguity in deep wate. Futue studies will be equied to detemine which of these A B Fig. 7. Phonotaxic esponse pathways of epoductive female midshipman fish with ablated and sham-ablated lateal line systems. Positive esponse pathways of females with ablated lateal line systems (A) and sham-ablated lateal line systems (B) ae coloed geen and negative esponses ae coloed ed. The axes ae the distance fom the cente of the tank (cm) whee the monopole AQ 339 speake (black cicle) was located, and the dotted line epesents the position of the opaque sceen. Gey cicles indicate the position of the plastic mesh cylinde whee the animals wee eleased. 2083

7 (2014) doi: /jeb altenatives is coect and to empiically assess utilization of the diectional cues of acoustic pessue and adial paticle velocity duing sound souce localization by midshipman and othe fish. Contibution of the lateal line to sound souce localization Fish possess two mechanosensoy systems, the ea and the lateal line, which espond to many of the same stimulus fields (e.g. Coombs et al., 1989; Schellat and Wubbels, 1998; Baun and Coombs, 2000). While ealy theoists suggested that the lateal line was sufficient fo souce localization in the nea field, moe ecent studies demonstate that vibating dipole souces elicit physiological and behavioal esponses mediated by both sensoy systems, suggesting that eithe system may be sufficient fo souce localization (Van Begeijk, 1967; Nauoth and Mogdans, 2009; Baun and Coombs, 2010; Coombs et al., 2010). Single-unit ecodings fom tunk lateal line affeents in plainfin midshipman demonstate esponses up to 100 Hz, showing that tunk SN ae capable of encoding the Hz stimulus used in the pesent expeiments (Weeg and Bass, 2002). Howeve, the question of whethe the lateal line is equied fo localization behavio in this species had not been peviously exploed. In ou expeiments, animals wee eleased ~86 cm fom an 80 Hz sound souce, i.e. unde nea-field conditions in which both the inne ea and lateal line may be stimulated (e.g. Baun and Coombs, 2000). We obseved no statistical diffeence in sound souce localization by animals with sham-ablated lateal lines as compaed with those whee the lateal line was ablated with liquid N 2. These data thus suggest that the lateal line is likely not equied by this species fo souce localization in an axisymmetic sound field. Because we neve achieved 100% ablation, it is theoetically possible that the emaining neuomasts wee sufficient fo localization behavio. We conside this hypothesis unlikely because ablation was usually successful ove much of the animal, and the popensity fo positive phonotaxis did not appea to be elated to the popotion of suviving neuomasts (e.g. the animal with the geatest pecentage of suviving neuomasts, 17.6%, failed to localize the souce). Nonetheless, we cannot exclude the possibility that a few suviving neuomasts may be sufficient fo souce localization. Intact neuomasts wee most commonly detected along the vental mandible (see Table 1); thus, if the lateal line is used fo localization, the mandibula SN could be involved. Othe egions, such as the dosal tunk line, would not appea to be necessay, given that 100% of dosal tunk SN wee ablated in some fish that demonstated localization behavio (Table 1). Inteestingly, while females with ablated lateal lines demonstated positive phonotaxic esponses, thee was a significant diffeence in the mean oientation eo (elative to the sound souce) of the ablated vesus sham-ablated fish. These esults suggest a possible ole fo the lateal line in localization accuacy and imply complementay o synegistic oles fo the lateal line and inne ea in souce detection (e.g. Baun et al., 2002). Pehaps the lateal line is not indispensable fo localization in this context, but is instead used to fine-tune the appoach to the taget. In the mottled sculpin (Cottus baidii), the lateal line mediates oienting behavio to pey, and subtle diffeences in stike feeding wee noted in two pedatoy fish species (muskellunge, Esox masquinongy, and lagemouth bass, Micopteus salmoides) when the lateal line was inactivated with cobalt chloide (New and Kang, 2000; Coombs et al., 2001; Baun and Coombs, 2010). These studies povide behavioal evidence that the lateal line povides infomation impotant fo oienting behavios, consistent with the subtle diffeences in oientation to the sound souce we obseved in midshipman. Conclusions In this study, the plainfin midshipman fish was used as a geneal model to investigate the use of swim bladde pessue cues and lateal line mechanosensoy infomation in nea-field sound souce localization. Ou findings suggest that pessue eception via the swim bladde is likely impotant while the lateal line system may not be equied fo sound souce localization. Ou esults show that the midshipman can solve the 180 deg ambiguity in a complex field whee the potential diectional cues (phase elationship between pessue and paticle motion) ae the opposite to what they would be in a fee field and suggest, theefoe, that whateve stategy the female midshipman uses to coectly esolve the 180 deg ambiguity in ou test tank o in the extemely shallow nesting envionment would yield an incoect esolution in a deep-wate fee-field envionment. Theefoe, the question of how fish use acoustic pessue cues to solve the 180 deg ambiguity of souce diection fom the paticle motion vecto emains unesolved. MATERIALS AND METHODS Expeimental animals One hunded foty-six adult, epoductive female plainfin midshipman fish (Poichthys notatus) wee collected duing the summe midshipman epoductive season (May and June 2010). Fish wee collected fom the nests of Type I males (Sisneos et al., 2009) duing the moning low tides in the intetidal zone of Tomales Bay nea Mashall, CA, USA, which is the same geogaphical location used in pevious studies (Zeddies et al., 2010; Zeddies et al., 2012). Gavid females wee visually distinguishable fom Type I and II males based on the distended appeaance of the abdomen due to the pesence of eggs (Bass, 1996; Bass et al., 1999). Collected fish wee tanspoted in cooles with aeated seawate fom the field to the Bodega Maine Laboatoy (BML) in Bodega Bay, CA, USA. At BML, the fish wee maintained in lage communal tanks at natual ambient tempeatues (12 14 C) until late that night, when expeiments wee conducted. All expeimental pocedues wee appoved by the Univesity of Califonia Davis Institutional Animal Cae and Use Committee. Expeimental tank and setup All localization expeiments wee conducted at BML in an outdoo cylindical concete tank (4 m diamete, 0.75 m height), the same tank used in pevious studies (McKibben and Bass, 1998; McKibben and Bass, 2001; Zeddies et al., 2010; Zeddies et al., 2012). A sound souce (Lubell AQ339, Clak Synthesis, Littleton, CO, USA, o a US Navy J-9 tansduce) was suspended in the cente of the tank and positioned 10 cm above the tank floo. Note that two diffeent sound souces wee used fo these expeiments because the J-9 tansduce was unavailable fo the lateal line ablation expeiments. When using the AQ339, it was positioned with its adiating face oiented vetically. When using the J-9, the face of the pojecto was positioned such that it was oiented away fom the tank cente. A 2.44 m opaque plastic tap was placed immediately in font of, but not touching, the sound pojecto to pevent possible visual cues of the pojecto that might affect sound souce localization behavio. The acoustic playback signal consisted of a continuous tone that mimicked the fundamental fequency of the male advetisement call, as this stimulus elicits obust positive phonotaxis in gavid female plainfin midshipman fish (Bass et al., 1999; Bass and McKibben, 2003). The playback signal used was eithe 75 o 80 Hz, based on the ambient tempeatue of the wate in the tank, as pevious studies epoted that female midshipman showed a tempeatue-dependent fequency pefeence in thei phonotaxic esponses (McKibben and Bass, 1998). While tempeatue may also influence auditoy sensitivity in some fish species (Wysocki et al., 2009), in the pesent expeiment both contol and expeimental animals wee tested on a given night using the same tempeatue paametes, allowing fo compaisons between teatment goups. The acoustic playback stimuli wee geneated by an Agilent 33120A function geneato and passed though a Kohn-Hite 3550 filte ( Hz bandpass) to a powe amplifie (Cown Audio Inc., Elkhat, IN, USA) that dove the sound pojecto. Acoustic 2084

8 (2014) doi: /jeb pessue levels wee veified nightly pio to behavioal expeiments by placing a hydophone (model 8103, Büel and Kjae, Nocoss, GA, USA, o model BM8178-7, Sonatech, Santa Babaa, CA, USA) at one of the two sites within the tank whee fish would be eleased (Fig. 1). The tone level at the calibation site was set at 130 db (e. 1 μpa peak), consistent with sound pessue levels of the advetisement calls ecoded nea the nests of Type I males (Bass and Clak, 2003). The behavioal esponses of female midshipman fish wee ecoded using a digital video ecode and a CV110 Pecision black-and-white camea (0.2 lx minimum light level) mounted ~6 m above the outdoo test aena. The video ecodings wee digitized using a Vixia HV30 camcode (Canon) and imovie 7.0 softwae (Apple Inc., Cupetino, CA, USA). Windows Movie Make 5.0 (Micosoft, Redmond, WA, USA) and SigmaScan Po 5.0 (Systat Inc., Chicago, IL, USA) wee used fo fame-by-fame analysis of the digitized video ecods. Evey fifth fame was analyzed by making the position of the animal s head (on the midline between the two eyes) elative to the fixed position of the sound pojecto. The x and y coodinates of the animal s head wee then used to tack the movement of the animal in elation to the sound souce and sound field. Acoustic measuements In ode to chaacteize the fields poduced by the J-9 and AQ339 sound pojectos, acoustic pessue and paticle motion measuements wee made following pocedues descibed in pevious studies (Zeddies et al., 2010; Zeddies et al., 2012). Pessues wee measued using miniatue hydophones (Buel & Kjae model 8103), and paticle motions wee measued using a ti-axial acceleomete (PCB model W356A12; PCB Piezotonics, Inc., Depew, NY, USA) that was made neutally buoyant by embedding it in syntactic foam. Senso signals wee conditioned (Büel & Kjae model 2692 and PCB Model 482A amplifie fo the hydophones and acceleomete, espectively), digitized and stoed on a Windows-based compute. Using these sensos, the acoustic fields wee mapped in a hoizontal plane 1 cm below the souce cente. The scan egion spanned the font half of the test tank, whee all midshipman sound exposue expeiments wee conducted. Expeimental potocol Sound playback expeiments wee conducted between 21:00 and 05:00 h duing May and June Thee ed floodlights wee positioned aound the tank peimete, which allowed fo the obsevation and videotaping of the female midshipman phonotaxic esponses. The wate tempeatue in the test tank anged fom 10 to 12 C and was contolled by adjusting the incoming flow ate of seawate to the tank pio to the behavioal tests. Befoe testing began each night, the wate flow to the test tank was shut off and wate depth was adjusted to 50 cm, a depth typical of natual nests in the field. Female fish wee held individually in 5 gallon (=18.93 l) buckets filled with wate fom the test tank fo at least min pio to testing. Tests began when an individual fish was placed in a 30 cm diamete plastic mesh cylinde at eithe elease Site A o B inside the test tank, ~86.5 cm fom the sound souce (Fig. 1). Fish wee placed in the cylinde while the acoustic stimulus was continuously playing (no acclimation peiod). Tests wee teminated when the fish swam to the peimete of the testing aena o afte a positive phonotaxic esponse. A positive esponse was defined as the point when a fish diectly touched the speake face and/o cicled in font of o unde the sound pojecto. Although the opaque plastic tap visually obscued the position of the sound souce suspended above the tank substate, the fish was able to easily swim unde the tap in font of the speake to each the souce duing positive phonotaxis. Biased and unbiased elease expeiments In most expeiments, the mesh elease cylinde was open at the bottom to allow the fish to swim in any diection at onset of the test. In some expeiments, thee quates of the bottom cicumfeence was closed off, leaving a window fo the animals to exit that could be diected towad the sound souce. This biased elease was used to detemine whethe midshipman fish wee able to esolve the 180 deg ambiguity inheent in the paticle motion vectos pointing towad (and away fom) the sound souce: fish able to detect the axis of paticle motion but unable to esolve the 180 deg ambiguity would in some tials be expected to exit the cylinde away fom the souce in the unbiased (open) elease expeiments. Initial phonotaxis away fom the souce was physically impossible in the biased elease expeiments. Theefoe, if fish wee susceptible to the 180 deg ambiguity, 180 deg eos should have occued at a highe ate given an unbiased elease. Swim bladde deflation expeiments To investigate the necessity of the swim bladde fo sound souce localization, a goup of female midshipman undewent sugical swim bladde deflation. Fish wee fist anesthetized by immesion in a 0.025% benzocaine (ethyl p-aminobenzoate) seawate bath until opecula movement ceased. Afte the animal was anesthetized, a small (5 8 mm) incision in the body wall was made followed by anothe small (5 6 mm) incision in the swim bladde that allowed the bladde to be deflated. Swim bladde deflation sugeies wee pefomed on the day of animal collection, and completed 6 to 12 h befoe animals wee used in behavioal expeiments, which included a ecovey time of at least 3 4 h befoe expeiments wee pefomed. Veification of the swim bladde deflation sugeies took place within 12 h (the next moning) of the behavioal expeiments. Sham-deflated contol fish wee anesthetized and a simila small (5 8 mm) incision was made in the body wall along the tunk of the fish just dosal to the swim bladde at a position whee the bioluminescent photophoes ae found on the body. Following sugey, fish wee evived by flushing seawate ove the gills until opecula movement esumed and the fish showed nomal ighting behavio. Lateal line ablation expeiments The lateal line was selectively ablated with liquid nitogen. Fish wee anesthetized with a 0.025% concentation of benzocaine until opecula movement ceased. Small stainless steel o coppe wie pobes wee dipped in liquid N 2 and applied to the head and body of the fish to specifically ablate the supeficial neuomasts (SN) while the fish was unde deep anesthesia. To ablate the canal neuomasts (CN), canals wee fist opened with micodissection scissos, then the liquid N 2 -cooled pobe was applied and the canals wee e-sealed with supe glue (cyanoacylate). Fo shamablated contols, a subset of vental photophoes was selectively damaged with liquid N 2 using a pocedue othewise compaable to the SN ablation technique. Sham-ablated fish wee anesthetized fo the same peiod of time as lateal-line-ablated animals. The entie ablation pocess lasted min. Fish wee then evived by flushing seawate ove the gills until opecula movement esumed and the fish showed nomal ighting behavio. Ablated fish wee active upon ecovey fom anesthesia but did not show nomal behavioal escape esponses when gently stimulated with a wate jet applied to the skin, suggesting that the lateal line was not functioning. We noted that deep anesthesia (fish left in benzocaine fo a few minutes afte opecula movement ceased) was pefeable to shallow anesthesia (fish emoved fom benzocaine as soon as opecula movement ceased), as fish, afte shallow anesthesia, did not swim nomally in the hous afte ablation, eithe in the ecovey tank o in the sound localization test tank. Of the 50 animals that had ablated and sham-ablated lateal line teatment, 39 fish wee tested on the same day of sugey while 11 fish wee tested the day following sugey as a esult of the lethagic swimming esponse afte ecovey. Post hoc assessment confimed that the lateal line emained ablated in fish tested the day following the ablation pocedue, i.e. lateal line hai cells had not egeneated in the intevening <30 h. The lethagic esponse obseved in some animals tested shotly afte the ablation pocedue suggests a potential confounding effect of the benzocaine anesthesia and sugical ablation stess. Lateal line visualization Ablation success was qualitatively veified in a subset of animals immediately afte the behavioal localization expeiment. Fish wee immesed in 0.005% DASPEI [2-(4-(dimethylamino)styyl)-1-ethylpyidinium iodide; Life Technologies] in seawate fo 15 min in ode to visualize emaining neuomasts, insed in seawate, and anesthetized in a 0.025% benzocaine bath. Labeling was obseved using a Nikon AZ100 steeomicoscope micoscope equipped fo epifluoescence and images wee taken with a Coolsnap HQ2 2085

9 (2014) doi: /jeb camea (Photometix) and NIS Elements softwae. Afte imaging, animals wee euthanized with an ovedose of benzocaine, weighed, measued and fixed in 4% paafomaldehyde fo subsequent lateal line dissection and labeling with fluoescently tagged phalloidin. Fou egions of SN wee selected fo phalloidin labeling: a 2 cm egion fom the ight dosal line stating anteio to the dosal fin, a 1 cm egion on the top of the head (left side), cm fom the vental line (left side) stating at the anteio end of the anal fin, and the entie caudal fin. Given the small numbe of CN in this species, all canals wee dissected out of the head and labeled athe than selecting discete CN egions fo sub-sampling. Sampling egions fo SN and CN ae shown in Fig. 3. Dissected SN o CN wee teated with 20 μg ml 1 poteinase K fo 30 min at 37 C to digest the cupula and incease phalloidin penetation. Samples wee then incubated fo min in Oegon Geen phalloidin (Life Technologies) diluted 1:100 in 0.1 mol l 1 phosphate-buffeed saline (PBS), insed in fesh PBS, and mounted in 50% glyceol on bidged coveslips. Images wee taken on an Olympus FV-1000 confocal micoscope with associated Fluoview softwae. Confocal z-seies wee compessed into bightest-point pojections with ImageJ. Afte behavioal expeiments, the majoity of animals wee labeled with vital dye fo post-fixation visualization of the lateal line. These animals wee immesed in 3 μmol l 1 FM 1-43FX (Life Technologies) fo 90 s, insed in salt wate, and euthanized with an ovedose of benzocaine. Fish wee weighed, measued and then placed in 4% paafomaldehyde (in PBS) and stoed at 4 C. Fixed fish wee tanspoted back to the Univesity of Washington, insed in fesh PBS, and the labeled lateal line was visualized with a Leica MZFLIII steeomicoscope. Images wee taken with a Leica DFC350FZ camea and associated Fiecam softwae (v. 3.2). The numbe of emaining SN in the FM 1-43FX-labeled animals was quantified in six distinct egions of each animal: the ight dosal lateal line adjacent to the dosal fin, the SN cluste on the dosal head suface (ight side), the vental line stating anteio to the pelvic fins and extending to just behind the left pectoal fin, the vental-most line on the left opeculum, the mandibula line, and the entie caudal fin (see Fig. 2). These egions wee selected based on initial obsevations in DASPEI-labeled animals that ou ablation pocedue was highly effective in some lateal line aeas (e.g. the dosal suface of the head), while othe aeas wee moe difficult to access with the fozen pobe and theefoe contained moe suviving neuomasts (e.g. the mandibula line). Data analysis The effects of swim bladde deflation wee analyzed using logistic egession. Phonotaxis esponses wee categoized as 1 (coect localization) o 0 (incoect) and fit to a logit function using a maximum likelihood method (Menad, 2002). Logistic egession was also used to examine the effects of deflated swim bladdes as compaed with einflated and shamdeflated swim bladdes. To analyze the movement of the fish, the diffeence angles of the beaing of the fish elative to the souce and elative to the local sound field wee detemined. This was done by detemining the position of the fish fom the video ecod (at 150 ms intevals) and then calculating the fish s beaing between consecutive positional points. The diffeence angle elative to the souce was the diffeence between the fish s beaing and the angle fom the fish s position to the souce and was calculated between each time step (150 ms) fo all ecoded behavioal tacks. The diffeence between the fish s beaing and the diection to the souce was defined as the oientation eo. Statistical analyses wee pefomed to detemine whethe thee wee diffeences among the expeimental goups in the diffeence angles at two specific points in the phonotaxic pathway: (1) at the initial movement out of the elease point (as defined as the vecto between the fist obseved position outside the elease cylinde and the fifth obseved position) and (2) at the midpoint between the elease point and the speake. As a measue of pefomance, the vecto stength () of the diffeence angles was computed. Vecto stength is a measue of diectional tendency o consistency towad the souce (Batchelet, 1981); moe fomally, it is the nomalized length of the mean vecto of the cicula distibution of angles to the souce (the vecto fo each fish is unity length with the angle to the souce, o in line with the paticle motion vecto). If all diections ae equally likely, the vecto stength is zeo, wheeas if all fish move in the same diection, the vecto stength is 1.0. Diffeence angles wee tested fo unifomity using a Hodges Ajne test (Za, 1999), which tests the null hypothesis that these angles ae andomly distibuted. A significant P-value indicates a bias in the distibution of diffeence angles, i.e. the null hypothesis of equal (andom) distibution is false. Diffeences between teatments wee analyzed using a Watson Williams test (the cicula equivalent of a two-sample t-test) (Watson and Williams, 1956), which analyzes whethe two vectos have the same mean diection. The P-values wee Bonfeoni coected to α=0.025 to eflect the tests conducted at two points in the phonotaxic pathway. Though this test assumes that the data have an undelying von Mises unimodal distibution, the test is obust to deviations fom this assumption (Za, 1999). All tests wee conducted using the cicula statistics toolbox (Beens, 2009) fo MATLAB vesion 2009b (MathWoks, Natick, MA, USA). APPENDIX Resolving the 180 deg ambiguity The field of a point monopole in a fee-field is an achetype fo the diectionalization poblem. It is the simplest possible case, yet one that occus often in natue. It would be expected that any method that a fish might use to esolve the ambiguity would have to wok fo this case to be of geneal use. The mathematics can be simplified futhe by assuming that the signal is sinusoidal. This is a good appoximation fo the advetisement call of the midshipman and was exactly the case in ou expeiment. We focus hee on the diectionalization cues pesent in the stimulus, not on the algoithm employed by fish such as those poposed by Roges et al. (Roges et al., 1988) o Schuijf and Buwalda (Schuijf and Buwalda, 1975). Any poposed algoithm must exploit these cues. We assume, theefoe, that the fish can detect both the acoustic paticle velocity vecto (elative to its own body axis) and the acoustic pessue. We also assume that the discimination is to be made by simultaneously sensing both quantities at the same point in the wate. Fo a sinusoidal signal, the only paametes that can be measued ae the amplitude and phase of the two field quantities. Conside a point monopole souce, oscillating at angula fequency ω, located at the oigin of a spheical coodinate system. Using complex notation, the acoustic pessue at a distance fom the souce is given by: pt = pe ω = p e ik (,) Re[ˆ( ) i t 0 ] Re ˆ e iωt 0, (A1) whee ˆp() is the complex amplitude of the eceived acoustic pessue, ˆp 0 is the complex souce level of the souce, k (=ω/c) is the wavenumbe, 0 is some efeence distance and Re denotes the eal pat of the complex quantity in backets. Fom Eule s equation, the acoustic paticle velocity in the adial diection is given by: p i e v (,) t = Re[ˆ v ( ) e ω ] = Re ˆ ik i t ω ρ 1 e i t, (A2) c k whee ˆv () is the complex amplitude of the adial paticle velocity and ρ and c ae the density and speed of sound in wate, espectively. Fom Eqns A1 and A2, it is evident that the complex adial velocity is popotional to the complex acoustic pessue: 1 i vˆ ( ) = + ρ 1 p ˆ( ). (A3) c k The quantity in backets is the acoustic admittance, the invese of the acoustic impedance. The eal pat of the tem in backets is popotional to the component of the adial velocity that is in phase 2086