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Masters Theses & Specialist Projects Graduate School
11-2009 The evelopmeD nt and Role of Peripheral Auditory Structures in Otocinclus affinis Sri Kiran Kumar Reddy Botta Western Kentucky University, [email protected]
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Recommended Citation Botta, Sri Kiran Kumar Reddy, "The eD velopment and Role of Peripheral Auditory Structures in Otocinclus affinis" (2009). Masters Theses & Specialist Projects. Paper 128. http://digitalcommons.wku.edu/theses/128
This Thesis is brought to you for free and open access by TopSCHOLAR®. It has been accepted for inclusion in Masters Theses & Specialist Projects by an authorized administrator of TopSCHOLAR®. For more information, please contact [email protected]. THE DEVELOPMENT AND ROLE OF PERIPHERAL AUDITORY
STRUCTURES IN OTOCINCLUS AFFINIS
A Thesis
Presented to
The Faculty of the Department of Biology
Western Kentucky University
Bowling Green, Kentucky
In Partial Fulfillment
Of the Requirements for the Degree
Master of Science
By
Sri Kiran Kumar Reddy Botta
December 2009
THE DEVELOPMENT AND ROLE OF PERIPHERAL AUDITORY
STRUCTURES IN OTOCINCLUS AFFINIS
Date Recommended 23rd Nov 2009
Michael E. Smith_____
Director of Thesis
____ Claire A. Rinehart____
__ Nancy A. Rice______
______Dean, Graduate Studies and Research Date ACKNOWLEDGEMENTS
First of all, I like to convey loving gratitude to my parents. Without their support,
I may not be in the position where I am now. I would like to thank Dr. Michael E. Smith, who has always been friendly, helpful and supportive in many situations and made me understand the theme and complete my thesis on time. I am grateful to be graduating under his advising. I am thankful for his patience in thesis correction and with my language. I would also like to thank my graduate committee members Dr. Claire Rinehart and Dr. Nancy Rice for being kind and for their helpful comments on my thesis. I would also like to thank the Dean of the Graduate studies, Dr. Richard Bowker and all the
Biology departmental staff for their constant support.
I would like to thank Dr. John Andersland for helping me with microscopy and giving suggestions throughout my Master’s study. I like to thank to my lab mates, friends and other graduate students for their help and moral support. Last but not least, I like to thank Western Kentucky University for giving me pleasant memories during my stay in
Bowling Green.
Finally I would like to thank God for every thing he has given me so far.
i TABLE OF CONTENTS
PAGE CHAPTER 1….………………………………………………………………………..3-8
Introduction.…………………………………………………………………………….3
CHAPTER 2…………………………………………… …………………………....9-26
Introduction……………………………………………………………………………..9
Material and Methods….………………………………………………………………11
Results.…………………………………………………………………………………13
Discussion…..…………………………………………………………………………..23
CHAPTER 3…..……………………………………………………………………..27-44
Introduction….…………………………………………………………………………27
Material and Methods…..………………………………………………………………30
Results.…………………………………………………………………………………34
Discussion…..…………………………………………………………………………..41
REFERENCES..……………………………………………………………...... 45
ii LIST OF FIGURES
Page
1: Peripheral and inner ear auditory structures of an otophysan fish ……….………..…...7
2: Peripheral and inner ear of Pterygoplichthys gibbiceps …………………...…………..8
3: Development series of Otocinclus affinis used in this study .………………………...16
4: Live 5.25 mm TL O. affinis showing the skull region ……………………………….17
5: Compound pterotic bone in 7.5 & 12 mm TL O. affinis ..……………………..……..18
6: Dorsal and lateral view of 12 mm TL O. affinis ………….……….………………....19
7: Dorsal view and drawing of an adult O. affinis ……….....…….……..………………20
8: Lateral view and drawing of an adult O. affinis ………………………………..….....21
9: Pectoral spine connection to the compound pterotic bone in O. affinis ………….…..22
10: X-ray image of O. affinis with inflated and deflated swim bladder ….……………..36
11: Audiograms of O. affinis with inflated (control) and deflated swim bladders .……..37
12: Threshold shifts resulting from swim bladder deflation ………………...…………..38
13: Audiograms of O. affinis with non covered (control) and covered fenestrae ..……...39
14: Threshold shifts resulting from fenestrae covering ………….……………………...40
iii THE DEVELOPMENT AND ROLE OF PERIPHERAL AUDITORY
STRUCTURES IN OTOCINCLUS AFFINIS
Sri Kiran Kumar Reddy Botta December 2009 50 Pages
Directed by: Michael E. Smith, Claire A. Rinehart, and Nancy A. Rice
Department of Biology Western Kentucky University
Loricariidae is a very diverse family of catfishes found primarily in the Amazon
River basin. These catfishes have a unique characteristic feature of having fenestrae
(holes) in the skull region (compound pterotic bone) adjacent to their bi-lobed swim bladder. Since the swim bladders and the compound pterotic may act as an external ear for hearing in this taxon, I hypothesized that these swim bladders structures have an acoustical functional in the loricariid Otocinclus affinis. In order to understand the development of these structures in O. affinis, I first monitored the ontogeny of the compound pterotic bone by clearing and staining of fish ranging total length from 0.75 to
3.5 cm. the swim bladders and fenestrated compound pterotic bone were developed at early larval stages (by 5.25 mm TL). Second I examined the role of swim bladders in hearing in adult O. affinis, by testing the hearing sensitivity before and after swim bladder deflation. Hearing thresholds were determined electrophysiologically by recording auditory evoked potentials (AEP). Swim bladder deflation increased hearing thresholds by 19 – 23 decibels (dB). I then tested the role of the compound pterotic bone fenestrae in hearing by covering them with a tissue adhesive (n-butyl cyanoacrylate) and recording
iv the hearing thresholds. Covering fenestrae increased hearing thresholds by 4 – 11 dB.
Future experiments are required to more precisely determine the acoustical role of these peripheral auditory structures in O. affinis.
v CHAPTER 1
Introduction:
Since sound travels approximately five times faster in water than air, acoustic communication is a very effective way to communicate under water. Fishes have evolved the capability to both hear and generate sounds for communication. The auditory structures and hearing evolved in fishes several millions of years ago, and it is thought that the sense of hearing first evolved in fishes (Popper and Fay, 1998). As a result, the organs present inside the body of fish are homologous to those of present day mammals.
There is a considerable amount of variability in the auditory structures of fishes, but all fish ears are composed of pairs of three otolith-bearing end organs – the utricle, saccule, and lagena, as well as three semicircular canals. The inner ear consists of a membranous labyrinth divided into the pars superior (semicircular canals, utricle and ampullae) and pars inferior (sacculus and lagena) (Arratia, 2003). Sound waves travel through the fluid- filled sacs of the sacculus and lagena, they initiate movement of the otoliths present over the auditory sensory hair cells. These hair cells convert the vibration into an electrical signal and send the signal via afferent nerves of the auditory nerve to the hearing centers of the brain.
Some fish taxa have unique peripheral auditory structures that enable them to hear with a greater sensitivity over a broader range of frequencies (0.1 to 10 kHz) than other teleost fishes (Ladich & Wysocki, 2003; Lechner & Ladich, 2008). Some of these fishes have modified vertebral elements, which was discovered and named after the German anatomist Ernst Heinrich Weber in 1820 (Tavolga 1971). He not only described the inner ear but also showed that the ear was connected to the swim bladder through a series of
3 4 moveable bones (tripus, intercalarium, scaphium and claustrum) connected to one another by ligaments and then connected to fluid-filled sacs (sinus impairs and canalis transverses) adjacent to the inner ear. These series of bones are called the Weberian ossicles. The Weberian apparatus is one of the unifying characteristics of the fishes in the superorder Otophysi. This group contains majority of freshwater fishes, including catfishes, minnows, loaches, characids, gymnotids and knife fishes, etc.
In general, the density of a fish body is similar to the density of the surrounding water, so waterborne sound transmits directly through the fish body. Two exceptions to this are the swim bladder, which is filled with low-density gas, and the otoliths present in their ears, which are composed of a high-density calcium carbonate and protein matrix.
Sound reception under water requires a transducer that is different in density from the surrounding water. In many fishes, the gas-filled swim bladder serves as such a transducer (Tavolga, 1971). When sound waves impinge upon the swim bladder, it vibrates in response to the pressure wave. In otophysan fishes, this vibration is then transmitted through the Weberian ossicles - first through the tripus, then followed by the intercalarium, scaphium and clustrum, and then on to the fluid filled sac of the cavum
(sinus impar; Figure 1). These vibrations are then transferred to the endolymphatic fluid filled sac (canalis communicans) to the inner ear (Weitzman, 2005).
The order Siluriformes (catfish) is one of the largest groups in the superorder
Otophysi. They are found in saltwater as well as in fresh water, but the majority of them are freshwater species. This taxon currently consists of approximately 36 families and over 3100 known species (Ferraris, 2007) ranging in size from 3.5 centimeters to several meters. They are mainly classified on the basis of their osteology. Loricariidae is the 5 largest catfish family with almost 646 accepted species (Armbruster, 2003). They are mostly found in the tropical regions of South America especially in the regions of the
Amazon River basin. They are commonly known as armored suckermouth catfish because of the positioning of their non-closable mouth, which is surrounded by rectal barbels, which helps them to adhere to a substrate when exposed to strong current, and the elongated bony plates covering their bodies.
Another distinct morphological feature of these catfishes is that they possess a bi- lobed swim bladder instead of a large, free swim bladder that is found in most other catfishes. Loricariids also have many channel-like openings (fenestrae) in the bone that encapsulates the bi-lobed swim bladders. These fenestrae are filled with lipids, which then make contact to the outer layer of the swim bladders. Instead of having several
Weberian ossicles as is found in many otophysans, generally many loricariids have one or two ossicles attached to each swim bladder (Figure 2). These ossicles are believed to be a fused modification from the tripus and scaphium (Coburn & Grubach, 1998). These modifications of the swim bladder, and its surrounding bones, likely have some acoustical functionality. This morphological arrangement may assist sound entering the ear through the swim bladder or exiting the swim bladder from the vibrations produced by the pectoral spine.
Loricariids like many other catfishes have modified pectoral spines, which can be used as a defensive weapon but can also be used to produce. These enhanced pectoral spines are moved by abductor muscles (Ladich et al., 2006). The base of the pectoral spine has a series of ridges, which stridulates on the surface of the cleithrum producing a 6 series of short pulses (Ladich 1997a). Otocinclus affinis, a very small loricariid, have been shown to produce sounds in this manner (Amanda Webb, unpublished data).
Although it has been nearly 200 years since Weber discovered and explained the role of Weberian apparatus in fish hearing, there is still much that is not understood about the process in general. Hearing tests have been performed on relatively few taxonomy groups. For example, in Loricariidae the largest catfish family, hearing tests have been performed in only a few species (Lechner & Ladich 2008). Most teleost fishes have a free swim bladder, whereas the Loricariidae have modified peripheral auditory structures like a minimized Weberian apparatus, bi-lobed swim bladders and fenestrae in the swim bladder capsule bone adjacent to the bi-lobed swim bladders. How these modifications relate to their hearing sensitivity is not clear. Examining these structures and how they affect hearing in a loricariid species is the goal of this thesis.
The loricariid I chose for this study is Otocinclus affinis (golden or dwarf oto).
They are widely distributed in tropical freshwater rivers in South America and are generally small in size ranging from 3 to 4 cm total length (TL). They are found mostly in small streams or along the margins of larger rivers, clinging to substrates using their mouth as a sucker, and feeding from algae, stones, macrophytes, and broad-leaved grasses (Hans & Ingo, 2005). Just like all loricariids O. affinis have the special feature of having fenestrae in the compound pterotic bone that encapsulates their bi-lobed swim bladders. In fact, some Otocinclus refer to these lattice-like fenestrae (oto = ear in Latin, cinclus = latticework).
This thesis is divided into three chapters. Following this introductory chapter, I describe the morphology and development of the fenestrae at both larval and adult stages 7 of O. affinis (Chapter II). In chapter III, I present the method and results of two experiments that examine the role of the swim bladders and fenestrae in hearing of O. affinis.
Figure 1: Drawing of the swim bladder and the associated Weberian apparatus connected to the inner ears of the otophysan fish Carassius auratus (modified from Landford, 2000 and Tavolga, 1971). When the sound waves (arrows indicate the direction of sound waves) impinges on the swim bladder, the swim bladder vibrates in response to the wave and transmits the sound to the inner ear through a series of Weberian ossicles (tripus, intercalarium, scaphium and claustrum, colored orange) and the fluid filled canals (sinus impairs and transverse canal, colored green). 8
Figure 2: Outline of Pterygoplichthys gibbiceps inner ear and associated structures including swim bladders and Weberian ossicle. The bi-lobed swim bladders are connected to the inner through the modified single Weberian ossicle (t) and fluid filled canals (atium sinus impairs: asi and transverse canal: tc). Grayed areas inside endorgans indicate relative position of otoliths in the utricle (U), lagena (L) and saccule (S). This picture is modified from Rogers (2009).
CHAPTER 2
Introduction:
Catfishes of the family Loricariidae are recognized by an elongated body, compressed ventrally and generally covered with bony plates. The mouth is ventrally placed and is often modified into a sucking disc. A pair of maxillary barbels is present, connecting the upper lip and lower lip (Teugels et al., 2001). The skull region of loricariids differ from non-loricariids catfishes in three different aspects: 1) the presence of compound pterotic bone (comp-pt) instead of numerous bones; 2) the presence of fenestrae (channel-like holes) on the comp-pt bone; and 3) expansion of the dorsal portion of comp-pt bone to enclose the lateral opening of the swim bladder capsule. The fenestrae on comp-pt bone (posttemporosupracleithrum bone as per Schaefer 1997;
Arriata, 2003 or pterotic+supracleithrum as per Weitzmann, 2005) are present in all loricariids, but shape, size and distribution are variable among the species. These fenestrae are filled with lipids that form a barrier between the swim bladder and external skin of the fish. It is possible that these lipids act as an impedance matcher, which assists sound transmission either into or out of the swim bladder since the impedance of lipids are intermediate between that of the surrounding water and gas in the swim bladders.
Such impedance matching may prevent acoustical reflection at the interface between the swim bladders and the rest of the fish body.
In loricariid fishes the compound pterotic bone is found as a single compound bone but in non loricarioid fishes the comp-pt bone is present in the form a complex of four separate bones (postemporal, supracleithrum, extracapular and transcapular ligments) instead of a single bone (Adriaens & Verraes, 1998). Wright in (1885) stated
9 10
that the supracleithrum is fused to the pterotic in Hypophthalmus and loricariids. This
concept was controversial for nearly a century until Fink and Fink (1981) and Arratia and
Gayet (1995) confirmed that the posttemporal, supracleithrum and ossified Baudelots
ligments (extracapular and intracapular ligments) fused together to form a compound
bone. This bone is referred as the compound pterotic (comp-pt) in this paper.
In some catfishes, the compound pterotic bone remains moveably articulated to
the skull but the pectoral-spine is less firmly attached to the compound pterotic through
the pectoral girdle (Diogo et al., 2001). In Otocinclus these compound pterotic bones surround the dorsal and lateral portion of the swim bladder on either side. This region is modified in such a way that it may improve the efficiency of the swim bladder as a transmitter of sound (Weitzmann, 2005; Lechner & Ladich, 2008). These fenestrae are situated laterally over the swim bladders and are angled in such a way that they may focus acoustical energy to the swim bladder. Perhaps, the comp-pt and swim bladder act as a sort of external ear in this species.
To better understand the biology of these fenestrae, I examined the morphology of
the compound pterotic and its associated fenestrae during ontogeny. Specifically, I
examined the osteology of O. affinis at four developmental stages (5.25, 7.5, 12 & 35 mm
total length: TL), with an emphasis of the structures surrounding the swim bladder and
inner ears. 11
Materials and Methods:
Animals:
O. affinis were ordered from commercial suppliers and immediately transferred to the tanks (91.4 ! 30 ! 50.8 cm) in the animal care room at Western Kentucky University.
Fish varied in total length from 3.1 to 4.1 cm total length (TL). A total of eight fish were used for this study (five adults and three larvae). The three larval fish used in this study were collected from the tanks in the lab containing adult O. affinis. Although a rare occurrence in capitivity, these larvae were spawned in the lab in 2007. Collected larvae were – 5.25, 7.5 and 12 mm in TL. Fish were euthanized via an over exposure of tricaine methane sulfonate (MS-222), although most fishes used in this study died of natural causes.
Clearing and staining:
Fishes were cleared and stained using the protocol of Song and Parenti (1995).
After measuring the total length and mass of each fish, it was fixed using 10% formalin for 24 hours. Then the specimen was rinsed in distilled water for 15 minutes, 4 times or until there was no smell of formalin. Then the fish was placed in alcian blue solution
(10mg in 84.2% ethanol + 15.8% glacial acetic acid) for 24 hours for an adult fish and 2 hours for larval fish. Afterwards, specimens were transferred through a dehydration series of 95%, 75%, 50%, 30% ethanol for an interval of 2 hours each. Then the specimen was rinsed in distilled water until the specimen sank in water. Then the specimen was placed in a trypsin solution (1 gm in 30% sodium borate solution) at 37°C until it digested most of the tissue (for adult fish it took 4-5 days and 8 – 9 hours for larvae). The specimen was transferred to 0.5% KOH for about an hour and then transfered into a bone staining 12 solution for a day or until the bones stained purple for an adult fish. For the larval fish the specimen was transferred to the 0.5% KOH for about 10 min and in alizarin red solution for 15 minutes or until the bones were stained purple. The bone staining solution was prepared by adding alizarin red slowly to 0.5% KOH while stirring until the solution turns deep purple. Then the specimen was destained in bleaching solution (3 drops of hydrogen peroxide in 100ml 0.5% KOH) for about 15 minutes for adult fish and 1 minute for larval fish. The specimen was then rinsed for 30 minutes in changes of 0%, 25%, 50% and 70% ethanol and finally stored in 70% ethanol.
Light Microscopy:
Specimens were observed under a Leica MZ16 (Leica, Bannockburn, IL, USA) that was mounted on the motorized z-axis plate where the plate moves from one focal point to another. Auto Montage software (Syncroscopy, MD, USA) was used to capture a series of images at different focal points. The software produced a merged image by combining focused parts of different series of images.
I used Macnification software (Orbicule BVBA, Belgium – Europe) to measure the size of the fenestrae. From the light microscope image this software measures the size by calibrating the microscopic picture to the microscopic scale provided. 13
Results:
Larval morphology:
Inflated swim bladders were readily apparent posterior to the compound pterotic
(comp-pt) bone in the 5.25 mm larva (Figure 3a & 4). The skull region was transparent, so that all the pairs of otoliths were visible through the dorsal view of the skull. The parieto-supraoccipital (o-par-soc) bone was visible and partially developed at this stage.
The lateral pectoral fins were developed but the dorsal fins were not evident in this stage.
Unfortunately this specimen was almost completely disarticulated during clearing and staining, so it was impossible to fully study the osteology. The compound pterotic bone was visible in the photographs of the live 5.25 mm specimen (Figure 4). The comp-pt bone formed a conical structure on each side of the head forming open holes through which the otoliths of the utricle, lagena and saccule could be viewed.
Swim bladders were clearly visible in the 7.5 mm larva (Figure 3b) and they had moved anteriorly towards the compound pterotic bone compared to the 5.25 mm specimen. Skin appeared less transparent in this stage, with an increased number of melanophores (Figure 3b). The o-par-soc bone appeared to be large and covered much of the dorsal region of the skull. The (comp-pt) bone was visible but not completely developed in this stage. The fenestrae on the comp-pt bone were visible and formed a conical structure, opened laterally and pointed towards the inner ear medially (Figure 5a).
Both the pectoral and the dorsal fins were visible in this stage.
The swim bladders were located more anteriorly in 12 mm larva when compared to the previous two stages (Figure 3c). The comp-pt bone was clearly visible and thicker compared to the previous stage. The comp-pt bone formed a conical tube like structure 14 lateral to the inner ear, which exposed the otolith of the utricle region (the lapillus; Figure
5b). The outer edge of the conical tube (comp-pt bone) was fenestrated. These fenestrae appeared better developed when compared to those of previous stages. The o-par-sac bone was more developed compared to previous stages and occupied the majority of the skull region (Figure 6a). The pectoral-spine was thicker compared to early stages and attached to the cleithrum. The dorsal fins and the posterior region of the fish appeared to be well developed at this stage(Figure 6b).
Adult Fish Morphology:
The swim bladders of the adult fish appeared directly under the compound pterotic bone instead of posterior to its as in earlier development stages (Figure 3d). The comp-pt had numerous, relatively large fenestrae, which were localized inferiorly on the bone (Figure 7 & 8). The anterior process of the parieto-supraoccipital (os-par-soc) appeared to be long and pointed towards the nasal plate (Figure 7).
The mean (" SE) maximum diameter of the fenestrae (measured from four cleared and stained adult fishes) was 0.23 (" 0.01) mm. The mean (" SE) minimum and maximum fenestrae diameter ranged from 0.13 (" 0.02) to 0.41 (" 0.02) mm in an individual fish. All the fenestrae channels appear to be at an angular position pointing towards the anterior of the comp-pt bone and medially towards the swim bladder. Larger fenestrae were placed at the lateral and posterior of the bone while smaller fenestrae are positioned more medially and anteriorly (Figure 7 & 8).
There were only five infraorbital (in-or4,5) plates around the orbit. A single heavy rostral plate covered the tip of the snout, which was surrounded by three oral plates bordering the mouth and the postrosrtal plates (pr-pl) sensu stricto Schaefer 1997. 15
Between these plates lay the rostral plates (r-pl) and prenasal plates (prn-pl). A small prefrontal plate (prfr-pl) covered the posterior portion of the lateral ethmoid. Two cheek plates (ch-pl) were present on the either side superior to the operculum (op). The comp-pt bone was found attached to operculum (op) and cleithrum (cl). It appeared that there was an opening lateral to the comp-pt (Figure 8). The lateral line canals are clearly visible along the lateral bony plates of adult fish with lateral line canal pores exiting the surface of these plates. The lateral line meets the edge of the compound pterotic at the lateral channel opening (Figure 8). The pectoral spine was attached to the compound pterotic bone through the pectoral girdle interiorly (Figure 9).
16
5.25 mm TL
7.5 mm TL
12 mm TL
35 mm TL
Figure 3: Developmental series of Otocinclus affinis used in this study. Arrows indicate the posterior edge of the swim bladders a) 5.25 mm, b) 7.5 mm, c) 12 mm, d) 35 mm total length (TL). 17
SB
Figure 4: 5.25 mm total length O. affinis. The arrows are pointed towards the otolith of the utricle (the lapillus). The triangle is pointed towards the otoliths of the lagena and saccule (the asteriscus and sagitta)and the dotted circle shows the position of the compound pterotic bone forming the conical structure exposing the inner ear. The bi- lobed swim bladder (SB) appear as golden spheres posterior to the compound pterotic. 18
Figure 5: The compound pterotic bone of the a) 7.5 mm TL and b) 12 mm TL larvae. Fenestration on the compound pterotic is visible in both specimens (arrows pointing towards the edges of fenestrated comp-pt) in the medial portion of the conical compound pterotic bone. The otolith of the utricle (lapillus; surrounded by the dotted white line) can be seen in b. 19
eye
o-par-soc pectoral-spine 1212 mm mm TL TL
comp-pt 0.2 mm o-par-soc comp-pt
eye
eye pectoral-spine 12 mm TL cleithrum 0.2 mm
Figure 6: a) Dorsal and b) lateral view of the 12 mm TL O. affinis larvae. comp- pt: compound pterotic bone, o-par-soc: os parieto-supraoccipitale. 20
Figure 7: a) Dorsal view of a cleared and stained adult O. affinis and b) corresponding drawing showing specific bones. comp-pt: compound pterotic bone, o-fr: os frontale, o- par-soc: os parieto-supraoccipitale, pr-pl: postral plate, prfr-pl: prefrontal plate, r-pl: rostral plate. 21
Figure 8: a) Lateral view of a cleared and stained adult O. affinis and b) corresponding drawing labelingspecific bones. ch-pl: cheekplate, comp-pt: compound pterotic bone, o- fr: os frontale, in-or4: infraorbital4, in-or5: infraorbital5, op: operculum, o-par-soc: os parieto-supraoccipitale, o-sph: os sphenoticum. 22
Figure 9: Ventral view of the connection betwween the pectoral spine to the compound pterotic via the pectoral girdle in an adult O. affinis. 23
Discussion:
Loricariidae are a group of catfishes, which have unique morphological traits
when compared to other catfishes. Specifically, they have a fenestrated compound
pterotic (comp-pt) bone which cover their bi-lobed swim bladders. In this chapter, I
described the ontogeny of these fenestrae as found in cleared and stained specimens of O. affinis. The compound pterotic bone develops at a fairly early ontogenetic stage in O. affinis (at least by 5.25 mm TL). This study was the first to systematically examine the ontogeny of the comp-pt bone in loricariids, although Geerinckx et al, (2007) labeled the comp-pt as a fused bone in a 9.8 mm TL specimen of Ancistrus cf. triradiatus, but in a
7.0 mm TL specimen the bone was separated into the supracleithrum and pterotic bones.
In O. affinis, I observed the formation of the comp-pt in live larvae as small as 5.25 mm
TL. Further clearing and stainng studies are required in order to study the ossification process of the comp-pt bone in early ontogentic stages of loricariids.
I observed that the compound pterotic bone was present dorso-laterally in between the parieto-supraoccipitale (o-par-soc) and the cleithrum encapsulating the swim bladders. Shape of the comp-pt bone was similar to that of its comp-pt bone in Ancistrus cf. triradiatus (Geerinckx et al., 2006). The comp-pt bone in O. affinis was similar to the closest relative Otocinclus vestitus examined by (Geerinckx, 2007). The comp-pt fenestrae had a mean diameter of approximately 0.23 mm. Measurements of the fenestrae in other loricariids species have not been studied before, but upon visually inspecting the
relative fenestrae size in O. affinis compared to the fenestrae size of much larger loricariid fishes such as Hypostomus plecostomus and Ancistrus cf. triradiatus, O. affinis have relatively larger fenestrae. While numerous fenestrae on the comp-pt bone are 24
present in all loricariids so far examined, they vary in size, shape, distribution and degree
of orientation from species to species (Schafer, 1987 & 1997). So it is unclear if there are
any functional relationships explaining this variability. It is possible that absolute
fenestrae size may be constant but relative size (ratio of the fenestrae size to the body
size) decreases in larger loricariid species.
I identified a lateral channel opening at the posterior edge of the comp-pt bone,
which has been reported in a few loricariid species. In most Loricariidae, this area of the
swim bladder capsule is completely closed, but in certain genera (e.g. Trichomycterus,
Vandellia, Ochmacanthus, Tridens, Pseudostegophills, Ituglanis and Homodiaetus) the
swim bladder capsule forms a small opening laterally to the supracleithrum (Aquino &
Schafer, 2002). In a few groups of loricariids, like Hypoptopomatinae, where the supracleithrum is fused as part of the comp-pt, a lateral channel opening is formed at the extreme posterolateral corner of the comp-pt bone (Aquino & Schafer, 2002). In
Ancistrus, another loricariid genus, there exists laterophysic connection between lateral- line and swim bladder. The lateral line passes through the trunk canals to the lateral opening and then underneath the comp-pt bone, where it makes contact with the swim bladders in Ancistrus (Aquino & Schafer, 2002).
Generally, the lateral line system is sensitive to water flow, measuring particle motion of water, but in order to be sensitive to sound pressure, the lateral line has to form an otophysic connection between the lateral line system and swim bladder (Webb, 1998).
In butterfly fishes (Chaetodon) from the order teleosts have an laterophysic connection underneath the supracleithrum. Butterflyfishes have a direct or indirect laterophysic 25 connection making the lateral line sensitive to the sound pressure waves (Webb & Smith,
2000). Variation in the laterophysic connections results in variation in sound sensitivity.
From these studies, I hypothesize that the lateral channel opening in O. affinis may have some acoustical sensory function in stimulating the lateral line. Since there was a lateral line that passes from the mid-body to the entrance of the lateral channel opening
(Figure 8), it is likely that O. affinis has a similar laterophysic connection as that found in
Ancistrus.
It is clear that the pectoral spine is directly connected to the comp-pt bone internally through the pectoral girdle in O. affinis (Figure 9). The pectoral spine was loosely attached to the comp-pt bone through the pectoral girdle and ligmentous tissue
(Reis, 1998; Diogo et al., 2001). The pectoral spine has multiple functions in catfish namely in defense and sound production. The pectoral spine produces sound by stridulation of the ridges of pectoral spine on to the inner surface of the cleithrum. In O. affinis, it is possible that the pectoral spine, which is connected to the comp-pt bone, that encapsulates the swim bladder, can emit sound through the comp-pt fenestrae and swim bladders. In other words, these structures that are thought to be peripheral auditory structures (bi-lobed swim bladders, compound pterotic with lipid filled fenestrations) could theoretically assist in sound production as well as sound reception.
In summary, the compound pterotic bone is developed and fenestrated in very early ontogenetic stages in O. affinis, even before it surrounds the swim bladder. The swim bladder plays an important role in hearing and sound production in many species of fish. Since O. affinis have evolved unique modifications like (fenestration of the comp-pt, a bi-lobed swim bladder and a single Weberian ossicle), it is likely that the swim bladder 26 and the bones surrounding it have acoustical functionality in either sound production and/or sound reception. CHAPTER 3
Introduction:
The swim bladder is a gas-filled organ that is present in the abdominal cavity region in most fishes. The swim bladder wall is made up of three membranes, the tunica externa, submucosa, and tunica interna (Chardon, 2003). The swim bladder is known to have many functions. It acts as a hydrostatic organ, which aids in the control of fish density through varying gas pressure inside the bladders to help the fish regulate buoyancy (Steen, 1970). It can also serve as an acoustic transformer during sound production (Demski et al, 1973), which may serve as a means of communication. Teleost fishes can produce sound in a number of ways. For some species sound is produced by expulsion of gas from the swim bladder through the pneumatic duct. Other species have intrinsic muscles or sonic muscles along the swim bladder, which initiates vibration in the swim bladder wall to produce sound (Skoglund, 1961). Swim bladders not only assist in sound production, but can also act as a transducer to aid in fish hearing. Weber (1820) first suggested that swim bladders along with Weberian apparatus assist in hearing but for over a century there were many controversies on this theory. The work of Von Frisch
(1923) conclusively demonstrated that the Weberian apparatus indeed assists in conduction of sound energy from the swim bladder to the inner ear in otophysan fishes.
Fish considered “hearing specialist” are taxa with unique peripheral auditory structures that allow the fishes to have more sensitive hearing over a broader bandwidth.
Fishes which do not have such specializations, are referred to as “hearing generalists”. In otophysan hearing specialists, the swim bladder is loosely connected to the tripus of the
27 28
Weberian apparatus through fibers of dorsal ventral pillars (Chardon, 2003). Sound pressure from a sound wave in the water is converted into a fluctuating motion as the gas in the swim bladder is compressed & decompressed. This resonation is then transferred via vibration to the Weberian apparatus, which is then passed through fluid filled sacs into the inner ear of the fish (Tavolga, 1971). Such a system allows specialist fishes to hear far field sound from 100Hz to low kHz range (Yan et al., 2000). Hearing specialists have better hearing sensitivity than fishes that do not have connections between the swim bladder and ear (Von Frisch 1938; Fay & Popper 1974, 1975; Yan, 1998; Mann et al.,
1997). Swim bladder positioning, shape and size vary across taxa (single lobed, bi-lobed, etc.). Over evolutionary time, the shape and size of the swim bladder evolved in different taxa of fishes according to their own functional needs (Hora, 1937). In catfish, species with larger swim bladders hear better at higher frequencies than those with smaller swim bladders (Lechner & Ladich, 2008).
Deflating or removing the swim bladder in fishes ear result in a significant increase in hearing thresholds (Von Frisch, 1938; Pogendrof, 1952; Kleerekoper &
Roggenkamp, 1959; Popper & Fay, 1974, 1975). When the same experiments were carried out in Tilapia macrocephalus (a hearing generalist) no threshold shift was observed. These studies support the idea that the presence of swim bladder connection to the inner ear via the Weberian apparatus improves hearing sensitivity.
In the current study, I examined Otocinclus affinis from the family Loricariidae.
Loricariids have the unique feature of smaller bi-lobed swim bladders distributed to the right and left of the vertebral column, behind the cranium (Lechner & Ladich, 2008).
Otocinclus have hearing capabilities that are similar to that of other otophysan fishes 29
(Michael E. Smith et al., unpublished data). The reduction in size of swim bladders and the number of ossicles improves the sensitivity due to free movable ossicles (Chranilov,
1929). Loricariid catfishes also have a unique feature of having openings (fenestrae or holes or windows) on the compound pterotic bone, which encapsulates the swim bladder.
The fenestrae channels seems to be angled towards the swim bladders, and may be pointed towards the tripus, which is connected to the medial portion of the swim bladder
(Weitzman, 2005). We hypothesize that these fenestrae serve some acoustical function but the role of these fenestrae on hearing has not been previously examined.
In order to examine the functional role of the swim bladders and fenestrae of the compound pterotic bone in O. affinis hearing, I performed two experiments. In the first experiment, I performed hearing tests on the fish before and after swim bladder deflation.
In the second experiment, I performed hearing tests on fish before (control) and after covering the fenestrae with tissue adhesive. Hearing tests were done electrophysiologically by recording auditory evoked potentials from the fish. 30
Materials and methods:
Animal:
O. affinis were ordered from commercial suppliers and immediately transferred to tanks (91.4 ! 30 ! 50.8 cm) in the animal care room at Western Kentucky University.
These fish varied from total length from 3.1 to 4.1. A total of 20 fish were used for this study.
Experimental design:
For this study I performed two sets of experiments using electrophysiology method. In the first set of experiments, I used 12 fishes to study the role of swim bladders in hearing of O. affinis. I first X-rayed the fish to ensure that the fish had intact swim bladders. Then I conducted hearing tests on the fish with intact swim bladders (control).
Hearing tests were performed by using electrophysilogical technique of measuring auditory evoked potentials (AEP). Once the control auditory thresholds were obtained the fish were allowed to recover in tanks for 5-7 days. Then the swim bladders were deflated and X-rayed to ensure that the swim bladders were deflated. After being X-rayed the deflated fish were subjected to subsequent hearing tests.
In the second set of experiment I used 8 fish to study the role of the compound pterotic fenestrae in hearing in O. affinis. After obtaining hearing thresholds on control fish, they were allowed to recover for 1-2 days. Then the fenestrae were covered using a tissue adhesive and the hearing tests were performed on the same fishes.
31
Swim bladder Puncture:
First, the fish were X-rayed using a dental X-ray machine (Gendex model no: 46-
137660620, Gendex, IL, USA) located in the Dental Hygienic Clinic of Western
Kentucky University. The fish were individually anesthetized using tricaine methanesulfonate MS-222 a common fish anesthetic. The fish was then placed in a water filled Petri dishes and gas from their swim bladders was removed using a 29 gauge 1cc insulin syringe. As the gas bladder is present under the compound pterotic bone, the needle was inserted into the lateral opening present between operculum, compound pterotic bone and cleithrum, and pierced into the gas bladder. The air was slowly drawn out of the swim bladder on each side of the head. The fish were then X-rayed to ensure proper deflation. After being X-rayed the fish was transferred back to the lab to perform hearing tests.
Tissue Adhesive:
In order to close the fenestrae in the compound pterotic, I used 3M Vetbond.
Vetbond is a strong tissue adhesive consisting of n-butyl cyanoacrylate, which polymerizes in seconds in contact with the tissue. It is used in sealing wounds in veterinary medicine. I dried the top of the fish head, including the area of the compound pterotic bone, using tissue paper and carefully covered the fenestrae using Vetbond tissue adhesive and allowed the adhesive to harden for approximately 30 sec. Once the adhesive was hardened, the fish was placed into the hearing test tank in preparation for a second hearing test.
32
Auditory evoked potential technique:
The auditory evoked potential (AEP) technique was the method used to perform
hearing tests. This electrophysiological technique records signals from the brain in
response to auditory stimuli. The test subjects were anesthetized with MS-222 and then
wrapped inside a fine nylon mesh and clamped using a metal clamp, which was attached
to a metal rod that was fixed into a micromanipulator. The animal was then inserted into
a 19-L cylindrical plastic tank closed on one side and open ended on the other. This tank
was placed on a vibration free air table and the entire setup was enclosed inside a walk-in
sound attenuation room (Whisper Room Inc, Morristown, TN).The speaker used for
producing sound stimuli (University Sound UW-30) was placed on the closed end burried
in sand so that just the top of the speaker protruded from the sand. The test fish was
adjusted in such a way that the fish was placed 6 cm below the surface of the water level.
Stainless steel subdermal electrodes (27 ga Rochester Electro-Medical, Inc, Tampa, FL,
USA) were used to record auditory evoked potentials. A recording electrode was placed
1-2 mm into the dorsal midline surface of the fish approximately halfway between the anterior insertion of the dorsal fin and the posterior edge of the operculae, directly over the brainstem. The reference electrode was inserted 1-2 mm subdermally into the medial dorsal surface of the head between the anterior portion of the eyes and the ground electrode placed on the tail region of the test animal. All the electrodes were pressed firmly, but carefully so as to not to harm fish. These electrodes were fixed into the successive inputs in the headstage and preamplifiers (RA4LI and RA16PA, Tucker Davis
Technologies, Inc., Alachua, FL, USA). During AEP recording, hydrophone (calibrated 33
sensitivity of -195 dB re. 1 V/"Pa; #3 dB, 0.02-10 kHz, omnidirectional, GRAS Type
10CT, Denmark) was placed adjacent to the fish to monitor the sound stimulus.
Both sound stimuli presented and AEP waveform recording were collected using
SigGen and BioSig software running on a TDT auditory evoked potential workstation
(Tucker-Davis Technologies Inc., Alachua, FL, USA). Sounds were computer-generated via TDT software and passed through a power amplifier connected to the underwater speaker. Tone bursts had a 2 ms rise and fall time, were 10 ms in total duration, and were gated through a Hanning window. Responses to each tone burst at each SPL were collected using BioSig software, with 200 responses averaged for each presentation. The
SPLs of each tone were confirmed using the hydrophone and a GRAS Pistorphone Type
$@AC calibrator. Sound pressure level was attenuated in 5dB steps until no further recognizable AEP waveforms were produced. In AEP audiometry, the lowest stimulus level that elicits a repeatable waveform is commonly taken as the threshold based on visual inspection.
Water in the test tank was transferred from the fish’s holding tanks and was maintained at the same temperature 28$C in order to minimize stress to the fish during transferring to the experimental tank. The hearing threshold levels were determined for the frequencies of 0.1, 0.25, 0.4, 0.6, 0.8, 1, 1.5, 2, 3, 4, 5, 6, 8 and 10 kHz. After hearing tests, the mass and TL of the fish were recorded and then the fish was placed back in their holding tank, feed regularly, and allowed to recover.
ANOVA:
Statistical analysis was performed using SYSTAT (version 11, Chicago, IL). The
effects of swim bladder deflation and covering fenestrae on auditory threshold levels 34 were tested using separate ANOVA’s, with treatment and frequency as independent variables. ANOVA was also used to test for the effect of frequency on threshold shifts of inflated vs deflated swim bladder fish samples, and as well as on non-covered vs covered fenestrae fish. 35
Results:
Swim bladders puncture experiment:
The auditory thresholds of fish with inflated swim bladders (controls) were similar to the thresholds previously obtained for O. affinis in the laboratory (Michael E.
Smith, unpublished data). Hearing sensitivity was greatest between 0.4 kHz to 1 kHz with the lowest mean (#SE) threshold at 1 kHz (89.4 # 1.8 dB re 1"Pa) and the highest at 10 kHz (154.2 # 2.3 dB re 1"Pa). The audiogram exhibits a U-shape curve that is common in fishes. The results from the X-ray validation showed that the swim bladder punctures were successful. The inflated swim bladders appear to be darker and show a greater contrast to the skull bones when compared to the deflated swim bladders in (Figure 10).
Following swim bladder deflation, the lowest hearing mean (#SE) threshold levels were at 1.5 kHz (111.4 # 4.1 dB re 1"Pa) and the highest hearing threshold levels were at 6 kHz (152.75 # 3.40 dB re 1"Pa). Removal of gas from the swim bladders had significant effect on hearing thresholds (P<0.001; Figure 11). There was an increase in thresholds of approximately 19 – 23 dB across all frequencies, with no significant threshold shift differences across frequencies (Figure 12). In swim bladder deflated fish, thresholds above 6 kHz could not be recorded because the hearing thresholds at these frequencies were beyond the limits of the AEP equipment.
Fenestrae covered experiment:
The threshold levels for the pre-Vetbond covering fish (control) in this experiment were similar to the controls of the deflated swim bladder experiment. The mean (#SE) lowest and highest threshold levels for the covered fenestrae were at 0.6 kHz
(93.2 # 2.2 dB re 1"Pa), and 10 kHz (159.5 # 0.9 dB re 1"Pa), respectively. Covering the 36 fenestrae of the compound pterotic bone with the tissue adhesive resulted in significantly elevated thresholds (P< 0.001; Figure 13). Threshold shifts ranged from approximately 4 dB at 8 kHz to 11 dB at 5 kHz, although these differences across frequencies were not statistically significant (Figure 14). 37
Figure 10: Dorsal X-ray view of O. affinis with a) inflated and b) deflated swim bladders. The arrows point to the swim bladders. The gas filled swim bladders are darker with more contrat to the surrounding bones than the deflated swim bladders.
Figure 11: Mean (!SE) auditory thresholds for control (n=12) and swim bladder deflated (n=7) of O. affinis.
37
Figure 12: Mean (!SE) auditory threshold shift for each frequency tested for swim bladder-deflated O. affinis (n = 7). Threshold shift is calculated as the difference between mean pre-andpost-deflated thresholds.
38
Figure 13: Mean (!SE) auditory thresholds for control (n=8) and fenestrae-covered of (n=6) O. affinis.
39
Figure 14: Mean (!SE) auditory threshold shift for each frequency tested for the fenestrae-covered of O. affinis (n = 6). Threshold shift is calculated as the difference between mean pre-and post-fenestrae-covered thresholds.
40 41
Discussion:
The audiogram for control O. affinis is similar to those of the other otophysan species with highest hearing sensitivity occurring between 600 and 1500 Hz. When compared with a commonly tested otophysan, the goldfish (Carassius auratus), the hearing thresholds of O. affinis were higher by 10 – 20 dB (Fay, 1969; Kenyon et al.,
1998; Yan et al., 2000; Smith et al., 2006). These higher thresholds could be the result of a lower signal to noise ratio in auditory evoked potential (AEP) recording from such a small fish as O. affinis, or they could represent a real lower sensitivity due to the differences in the auditory apparati of these two species. In addition, due to differences in protocols, there is considerable lab-to-lab variation among hearing thresholds recorded for the same species.
Lechner & Ladich (2008) conducted experiments on different species of catfishes in order to study how size, shape of swim bladder, and Weberian ossicles effect hearing in catfishes. They studied catfishes with large, free swim bladders versus small bi-lobed swim bladders. O. affinis appears to be similar in hearing abilities to that of the other bi- lobed loricariid species (i.e., Anistrus ranunculus, Hypoptopoma thoracatum and
Hemiodontichthys acipenserinus), which are most sensitive at 1 kHz. O. affinis have higher threshold levels compared to Corydoras sodalis and Dianema urostriatum these two non-loricariid catfishes in the family Callichthyidae have a bi-lobed swim bladder and a single Weberian ossicle catfishes. O. affinis have higher thresholds when compared to catfishes with a large, single-lobed swim bladder and four Weberian ossicles.
From their study, it is clear that fishes with a large, free single-lobed swim bladder with four Weberian ossicles have better hearing sensitivity at higher frequencies 42
than fishes with bi-lobed swim bladder and two or one Weberian ossicle (Lechner &
Ladich, 2008). Proximity of the swim bladder to ear also has a role in fish hearing. In
general, the closer the swim bladder is positioned to the ear, the better the fish hears
(Yan, 1998; Yan & Curtsinger, 2000; Ramcharitar & Popper, 2004; Ramcharitar et al.,
2006). The swim bladders in O. affinis are smaller when compared to those of other
catfishes, because of their small overall size. When considering size ratio of the swim
bladders to the size of the fish, O. affinis has relatively larger swim bladders, positioned
very near to the inner ear. O. affinis has only one Weberian ossicle present, similar to the
sensitive callichthyids. From these studies, I hypothesize that proximity of the bi-lobed swim bladder to the inner ear and their relative size are adaptations to improve hearing in
O. affinis.
Following the swim bladder deflation, auditory thresholds were 19 – 23 dB greater over all frequencies. When the swim bladder of Carassius auratus was deflated there was a threshold shift of 35 – 55 dB (Yan et al, 2000). Similarly when the swim bladder was removed surgically in goldfish there was threshold shift of 20 – 35 dB (Fay
& Popper, 1974). In channel catfish, Ictalurus punctatus, deflation of the swim bladder showed a significant increase in thresholds (Fay & Popper, 1975). These studies show
that the swim bladder plays a vital role in otophysan fish hearing. The removal of gas
from the swim bladders of Gadus morhua, a non-otophysan, resulted in increased hearing
thresholds (Sand & Enger, 1973). The cod fish have a modified swim bladder horn that
projects anteriorly, close to the ear. In contrast, there was no threshold shift when the gas
was removed from the swim bladders of non-otophysan fishes such as Trichogaster 43 trichopterus and Opsanus tau. This indicates that the swim bladder does not contribute significantly to hearing in these two non-otophysan species.
These studies show that even though there is the presence of a swim bladder in most fishes, it only assists in hearing of fishes with internal connections between the swim bladder and inner ear through the Weberian apparatus or other modifications. In O. affinis, the bi-lobed swim bladder is connected to the inner ear through a Weberian ossicle. The swim bladder deflation experiment showed that the swim bladder plays a definite role in hearing in O. affinis.
When the fenestrae of comp-pt bone were covered with VetBond in O. affinis, there was an increase in thresholds over all frequencies tested. A similar experiment was carried out with Botia modesta, which has a partial bony encapsulation of the swim bladder, with an open channel between the swim bladder and the skin of the fish
(Kratochvil & Ladich, 2000). In that study, the channel openings were covered with cotton and VetBond and sealed. Hearing tests were then conducted on the fish. This blockage resulted in a significant threshold shift over all frequencies tested. Normally the channel openings are filled with fluids, which may help the fish in hearing. It is not known whether these fluids are lipids similar to the lipids found in the fenestrae of O. affinis.
Lipids are known to form acoustic channels in cetaceans. For example, toothed whales (Odontoceti) have a lipid filled channel in their lower jaw which is connected to the inner ear. This may allow them to hear high frequency sounds (Koopman et al.,
2006). The fenestrae in O. affinis have lipid filled channels (Rogers, 2009), which may act as an impedance matcher (the process of transferring the energy from the external- 44 surface to the internal surface with minimal energy loss) between the external sound waves and the swim bladders. No previous studies have attempted to understand the function of the fenestrae of the comp-pt bone in loricariids. The increased thresholds from covering the fenestrae, suggests that they have an effect on hearing sensitivity in O. affinis. More direct measurements such as laser vibrometry studies are required to measure the effect of sound vibrations on the lipids inside the fenestrae channels.
Computer simulations modeling sound traveling through the fenestrae on the comp-pt bone and into the swim bladder may also be helpful in understanding the acoustical parameters of these fenestrae.
From these experiments, I conclude that the swim bladders and fenestrae on the compound pterotic bone have some acoustical function and play a role in O. affinis hearing. But further experimentation is required to fully understand the role of the fenestrae. It appears that, the smaller bi-lobed swim bladder and the single Weberian ossicle in O. affinis are compensated for, by relatively larger swim bladder volume for their small body size, proximity of the swim bladder to the inner ear, and relatively large fenestrae on the compound pterotic bone. This might be the reason for enhanced hearing in O. affinis compared to the other loricariids catfishes.
REFERENCES
1. Adriaens D. & Verraes W. 1998. Ontogeny of the osteocranium in the African
catfish, Clarias gariepinus (1822) (Siluriformes: Clariidae): ossification sequence
as a response to functional demands. J. Morph. 235: 183-237.
2. Aquino AE, Schaefer SA. 2002. The temporal region of the cranium of loricarioid
catfishes (Teleostei: Siluriformes): Morphological diversity and phylogenetic
significance. Zool. Anz. 241: 223–244.
3. Armbruster JW. 2003. Phylogenetic relationships of the suckermouth armoured
catfishes (Loricariidae) with emphasis on the Hypostominae and the Ancistrinae.
Zoo J Linn Soc. 141: 1-80.
4. Arratia G. & Gayet M. 1995: Sensory canals and related bones of tertiary
siluriform crania from Bolivia and North America and comparisons with recent
forms. J. Vert. Paleo. 15: 482–505.
5. Chranilov NS. 1929. Beiträge zur Kenntnis des Weber’schen Apparates der
Ostariophysi. 2. Der Weber’sche Apparat bei Siluroidea. Zool. Jahrb. Anat. 51:
323-462.
6. Coburn MM, Grubach PG. 1998. Ontogeny of the Weberian apparatus in the
armored catfish Corydoras paleatus (Siluriformes: Callichthyidae). Copeia 1998:
301-311.
7. Demski L, Gerald GW & Popper AN. 1973. Central and peripheral mechanisms
of teleost sound production. Amer. Zool. 13:1141-1167.
8. Diogo R, Oliveira C. & Chardon M. 2001. - On the osteology and myology of
catfish pectoral girdle, with a reflection on catfish (Teleostei: Siluriformes)
45 plesiomorphies. J. Morph. 249: 100-125.
9. Evers HG, Seidel I. 2005. South American Catfishes of the Families Loricariidae,
Cetopsidae, Nematogenyidae and Trichomycteridae. Catfish Atlas Vol. 1
Washington DC: Baensch.
10. Fay RR & Popper AN. 1974. Acoustic stimulation of the ear of the goldfish
(Carassius auratus). J. Exp. Biol. 61: 243-260.
11. Fay RR. (1969). Behavioural audiogram for the goldfish. J. Aud. Res. 9: 112-21.
12. Fay RR and Popper AN. 1975. Modes of stimulation of the teleost ear. J. Exp.
Biol. 62: 379-387.
13. Ferraris CJ Jr. 2007. Checklist of catfishes, recent and fossil (Osteichthyes:
Siluriformes), and catalogue of siluriform primary types. Zool. Taxa. 1418: 1-628.
14. Fink SV & Fink WL. 1981. Interrelationships of ostariophysan fishes (Teleostei).
Zoo. J. Linn. Soc. 72 (4): 297-353.
15. Geerinckx T, Brunain M, Adriaens D. 2005. Development of the chondrocranium
in the suckermouth armored catfish Ancistrus cf. triradiatus (Loricariidae,
Siluriformes). J. Morph. 266: 331–355.
16. Geerinckx T, Brunain M, Herrel A, Aerts P & Adriaens D. 2007. A head with a
suckermouth: a functional-morphological study of the head of the suckermouth
armoured catfishes Ancistrus cf. triradiatus (Loricariidae, Siluriformes). Belg. J.
Zool. 137 (1): 47-66.
17. Geerinckx T. 2007. Ontogeny and functional morphology of a highly specialized
trophic apparatus: A case study of neotropical suckermouth armoured catfishes
(Loricariidae, Siluriformes). Thesis desertation. 18. Hora SL. 1937. Correlation between the disposition of the liver and the kidney
and the form of the airbladder in certain siluroid fishes of India. Proc. Natl. Inst.
Sci. India. 3: 31-43.
19. Kenyon TN, Ladich F & Yan HY. 1998. A comparative study of hearing ability in
fishes: the auditory brainstem response approach. J. Comp. Physiol. A. 182: 307–
318.
20. Kleerekoper H. & Roggenkamp PA. (1959). An experimental study on the effect
of the swimbladder on hearing sensitivity in Ameiurus nebulosus (Lesueur). Can.
J. Zool. 37: 1-8.
21. Koopman HN, Budge SM, Ketten DR & Iverson SJ. 2006. The topographical
distribution of lipids inside the mandibular fat bodies of odontocetes: Remarkable
complexity and consistency. IEEE J. Oceanic Engineering. 31(1): 95-106.
22. Kratochvil H. & Ladich F. 2000: Auditory role of lateral trunk channels in
cobitids fishes. J. Comp. Physiol. A186: 279–285.
23. Ladich F, Bass AH. 2003. Audition. In: Catfishes. Vol. 2 Arratia et. al 2003. pp
701-730.
24. Ladich F, Wysocki LE. 2003. How does tripus extirpation affect auditory
sensitivity in goldfish. Hear. Res. 182: 119-129.
25. Ladich F. 1999. Did auditory sensitivity and vocalization evolve independently in
otophysan fishes Brain. Behav. Evol. 53: 288-304.
26. Ladich F. 1997a. Comparative analysis swimbladder (drumming) and pectoral
(stridulation) sounds in three families of catfishes. Bioacoustics. 8: 185-208.
27. Lanford PJ, Platt C, and Popper AN. 2000. Structure and function in the saccule of the goldfish (Carassius auratus): A model of diversity in the non-amniote ear.
Hear. Res. 143: 1-13.
28. Lechner W, Ladich F. 2008. Size matters: diversity in swimbladders and
Weberian ossicles affects hearing in catfishes. J. Exp. Biol. 211: 1681-1689.
29. Mann DA, Lu Z, & Popper AN. 1997. A clupeid fish can detect ultrasound.
Nature. 389: 341.
30. Poggendorf D. 1952. Die absolute Hörschwelle des Zwergwelses (Amiurus
nebulosus) und Beiträge zur Physik des Weberschen Apparates der
Ostariophysen. Z. Vergl. Physiol. 34: 222-257.
31. Popper AN & Fay RR, 1999. The Auditory Periphery in Fishes. In: Comparative
hearing: fish and amphibians. Fay RR & Popper AN. New York: Springer. pp 43-
100.
32. Ramcharitar JU. & Popper AN. (2004). Masked auditory thresholds in sciaenid
fishes: a comparative study. J. Acoust. Soc. Am. 116: 1687-1691.
33. Ramcharitar JU., Higgs, DM. & Popper AN. (2006). Audition in sciaenid fishes
with different swim bladder-inner ear configurations. J. Acoust. Soc. Am. 119:
439-443.
34. Reis RE. 1998. Anatomy and phylogenetic analysis of the neotropical callichthyid
catfishes (Ostariophysi, Siluriformes). Zool. J. Linn. Soc., 124: 105-168.
35. Rogers B. 2009. Morphology of the inner and peripheral ear of loricariid catfish
Pterygoplichthys gibbiceps K. Honors thesis Western Kentucky University. 37 pp
36. Sand O & Enger S. 1973. Evidence for an auditory function of the swimbladder in
the cod. J. Exp. Biol. 59: 405 - 414. 37. Schaefer S. 1987. Osteology of Hypostomus plecostomus with a phylogenetic
analysis of the loricariid subfamilies (Pisces: Siluroidei). Contrib. Sci. 394: 1-31.
38. Schaefer S. 1997. The Neotropical cascudinhos: Systematics and biogeography of
the Otocinclus catfishes (Siluriformes: Loricariidae). Proc. Acad. Natl. Sci.
Philad. 148: 1-120.
39. Skoglund CR. 1961. Function analysis of swim-bladder muscles engaged in sound
production of the toadfish. J. Biophys. Biochem. Cyt. 10: 187-200.
40. Song J, Parenti LR. 1995. Clearing and Staining Whole Fish Specimens for
Simultaneous Demonstration of Bone, Cartilage, and Nerves. Copeia. 114-118.
41. Smith ME, Coffin AB, Miller DL & Popper AN. 2006. Anatomical and function
recovery of the goldfish (Carassius auratus) ear following noise exposure. J. Exp.
Biol. 209: 4193-4202.
42. Steen JB. 1970. The swim bladder as a hydrostatic organ. In: Fish Physiology.
Vol. 4. Hoar WS and Randall DJ. New York: Academic Press.
43. Tavolga WN. 1971. Sound production and detection. In: Fish Physiology. Vol. 5.
Hoar WS and Randall DJ. New York: Academic Press. pp 135-205.
44. Tavolga WN. 1977. Mechanisms for directional hearing in the sea catfish Arius
felis. J. Exp. Biol. 67: 97-115.
45. Teugels GG, Sudarto & Pouyaud L. 2001. Description of new Clarias species
from Southeast Asia based on morphological and genetical evidence
(Siluriformes, Clariidae). Cybium 25: 81-92.
46. Von FK. 1923. Ein Zwergwels, der kommt, wenn man ihm pfeift. Biol. Zentralbl.
43: 439-446. 47. Von FK. 1938. The sense of hearing in fish. Nature 141: 8-11.
48. Webb JF. 1998. The Laterophysic connection: a unique link between the swim
bladder and lateral line system in Chaeetodon (Perciformes: Chaetodontidae).
Copeia 4: 1032-1036.
49. Webb JF & Smith WL. 2000. The laterophysic connection in chaetodontid
butterflyfish: morphology variation and speculations on sensory function. Phil.
Trans. R. Soc. Lond. B. 355: 1125-1129
50. Weber EH. 1820. De Aure et Auditu Hominis et Animalium. Pars I. De Aure
Animalium Aquatilium. Leipzig: Gerhard Fleischer.
51. Weitzmann SH. 2005. Hearing in catfishes especially that of the family
Loricarridae. In: Catfish Atlas. Vol. 1. Hans-Georg E, Seidel I. Washington DC:
Baensch. pp 31-39.
52. Wright RR. 1885: On the skull and auditory organ of the siluroid Hypophthalmus.
Trans. Roy. Soc. Canada 4: 107–118.
53. Yan HY & Curtsinger WS. 2000. The otic gasbladder as an ancillary structure in a
mormyrid fish. J. Comp. Physiol. A 186: 595-602.
54. Yan HY, Fine ML, Horn NS & Colon WE. 2000. Variability in the role of the
gasbladder in fish audition. J. Comp. Physiol. A186: 435–445.
55. Yan HY. 1998. Auditory role of the suprabranchial chamber in gourami fish. J.
Comp. Physiol. A 183: 325-333.