THE ORIGIN AND DEVELOPMENT OP THE GAS BLADDER IN THE GREEN SUNFISH, LEPOMIS CYANELLUS RAFINESQUE
Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy In the Graduate School of The Ohio State University
By ARTHUR EDWARD DUWE, B.S., M.S. i 1 * * The Ohio State University 1953
Approved by:
Adviser 1-
TABLE OF CONTENTS
Acknowledgments ------2. Introduction ------3 Historical Review ------5
Materials and Methods ------18 Observations ------22 Discussion ------32
Summary ------38 Abbreviations used on figures ------)p Figures ------lj.3,
Bibliography ------95 Autobiography ------99
A 16031 -2
ACKNOWLEDGMENTS
I owe a debt of gratitude to my wife Nancy and our two boys for their consideration during the time I have been working on this problem* I further wish to thank Nancy publicly for her assistance in editing and for typing the manuscript*
I wish to gratefully acknowledge the helpful suggestions and criticisms of my adviser Dr. John W, Price of the Ohio State University Department of Zoology. I want to thank also those of my colleagues who aided me in collecting material and gave me continuous moral support* - 3 - INTRODUCTION
It has been noted in the literature that some closely related species of fishes have different origins of gas bladders. The literature also dis closes that closely related species may have structurally different definitive gas bladders. Among some related fishes one species may possess a gas bladder while another lacks it* For these reasons and after having studied gas bladder origin and development in the bluegill the author decided to make a similar study on a species closely related to the bluegill. The green sunfish, Lepomis cyanellus Rafinesque, was chosen for this investigation because it too is in the genus Lepomis. and because of its availability in Central Ohio. Does the gas bladder In Lepomis cyanellus have an origin and development similar to that found in Lepomis macrochirus ? What are the circulatory ele ments and innervations of the gas bladder in Lepomis cyanellus? These questions can only be answered with certainty by direct Investigation. The green sunfish is a highly evolved and specialized Centrarchid and therefore the mode of -4- origin and development of Its gas bladder Is not necessarily predictable on a priori grounds. As in other groups, the pattern may conform to that of macrochlrus. but it may differ. In the latter case, differences between the two species at this point may or may not have evolutionary or phylogene tic significance, but at least should prove interest ing. -5- HISTORICAL REVIEW The gas bladder has been a subject of research
for over two hundred years. It is of interest to note that Needham as early at 1667, in "Disquisitio Anatomica”, published his idea that the gas bladder performs, in some fishes, some other function than that of a "float".
Quekett, (iBl^ij.), divided the general types of gas bladders Into three classes, "1. Those which are simple musculo-membranous sacs, having no duct of communication with the alimentary canal, or in fact any external opening. This kind of bladder Is found In the cod tribe, in the gurnard, sword fish, haddock, perch, and many other fish* 2. Those that are single, but have a duct of communication which is termed ductus pneumaticus or air duct. This opens sometimes into the stomach or oesophagus or some other part of the alimentary canal. Instances of this kind are found In the pike, salmon, sturgeon, trout, and many others. 3« Those which are double, the two compartments being kept together, or rather communicating with each other by a narrow tube, and the ductus pneumaticus in all cases proceeding from the posterior compartment. We have numerous examples of this kind of air bladder in most of our fresh - 6— water fishes, as the carp, barbel, tench, roach, chub, and gold fish,” Vogt, In l8ij.2, studied the development of Coregonus palaea and determined that the gas bladder originates from the esophagus as a solid mass whose cells are at first very much like those of the intes tine. Within this mass a cavity later appears which establishes communication with the esophagus. Price, (1935), states that In Coregonus clupeaformls the gas bladder does not arise until after hatching. In a later personal communication Dr. Price stated that in post hatch larvae of this species the gas bladder arises as a dorsal diverticu lum from the foregut. This observation agrees with that of the present writer as seen in the rainbow trout. The above citations Illustrate the variations in the mode of origin of the gas bladder that may occur within a single genus, i.e., Coregonus. According to Houghton, (1 8 6 8 ), the gas bladder is absent in the Cirrostomi and Cyclostomi, like wise It Is absent In the Holocephali and Plagiostomi.
The Protopteri, to which belong the Lepidosiren, possess a double air bladder, cellular and lung like, with air duct, glottis and pulmonary vein. -7- In Malacopterl a swim bladder and air-duct are both present, whereas some species of Anacanthini exhibit an air-bladder but no ductus pneumaticus. The Ganoidei possess, in some of the genera of the order, a lung-like structure which in some forms is cellular and provided with an air-duct,
Houghton also states that, "with regard to the existence of this organ in the different orders, it must be borne in mind that it is occasionally absent in fishes belonging to different genera, and some times even in fishes exhibiting merely specific differences", Morris, (1885), describes the Teleostean air- bladder as being very irregular in occurrence and character. When present in Teleosts it exhibits an extraordinary variation in shape, size, and posi tional relationship even between closely related genera and species. The air-bladder may have an open pneumatic duct, connecting with the oesophagus, or in a few cases with the stomach. Some Teleosts retain the duct but its cavity is closed. In some cases the duct is reduced to a fine filament while others have no trace of it. In the words of Houghton, "embryological evi dence indicates that an original function of the -8- air-bladder was the introduction of external air into the body, a function which has now lost its importance. And the apparatus for compressing and dilating the bladder may have been originally developed as an aid in this function. Also the extraordinary development of retia mirabilia, in the inner tunic of many air-bladders, now used only to secrete gas into the interior, may be a survival of ancient pulmonary capillaries, which have changed their character with their function". Houghton further points out that embryology points back to the condition of the primal fishes. Two groups of these primal fishes exist today in the Ganoids and Elasmobranchs. It is interesting to note that the Elasmobranchs are entirely devoid of a gas bladder, while all Ganoids possess it and also retain a fully developed pneumatic duct in the mature stage. In the suborder of Dipnoi the gas bladder is functionally active as a lung.
Tracy, (1911), uses the terms Physostomi and
Physoclisti in referring to the fishes having dif ferent types of gas bladders, the Physostomi being those with an open type gas bladder or those that retain the pneumatic duct, the Physoclisti being -9- those which have the closed type or those in which the duct is lost in adult stages. Kerr, (1919)» makes the following generaliza tions about the gas bladder: ”1* The primitive condition was that of a lung, communicating with the pharynx by a ventrally-placed glottis - for we have seen that the embryonic rudiment of the organ in the most archaic forms possessing it is a typical lung rudiment. 2. The organ became bilobed, growing back into a right lung and a left lung. 3 « In the forms which took to a purely swimming existence, and be came specialized in the direction of adaptation to this, there came about an asymmetry of the lungs, the right lung increasing and the left lung diminish ing. Lj.. In purely aquatic creatures the dictates of adaptation would naturally cause the air-filled lung to assume a dorsal position. 5. In the Actinop- teryglans, those fishes which show the highest degree of evolution In adaptation to a swimming mode of life, only the original right, lung persists as the air bladder. 6 . Finally In the Physoclistous forms - the most highly specialized of all - the swim bladder has become completely Isolated from the gut, Its respiratory function has gone and It subserves a mainly hydrostatic function”. -10- The above six points represent a scheme of evolu tion which seems probable* There exist details how- over which are still difficult to explain*
Hall, (l92ij-), gives the following general des cription of the gas bladder. ,rThe swimbladder, or air-bladder, of a fish, is situated dorsal to the coelom, between the alimentary canal and the verte bral column. It is a membranous sac containing the atmospheric gases. On a portion of the surface of
the bladder there is a vascular and glandular area, known as the rate mirabile, or T,gas gland”, or "red gland". Embryologically the swimbladder arises as a diverticulum from the dorsal side of the alimentary
canal. The primitive connection may be permanently
retained as a tubular canal, called the ductus pneu maticus, or it may be entirely absent in the adult*
Because of this variation the teleostean fishes have
been grouped into two divisions - the Physostomi,
characterized by the retention of the duct, and the
Physoclisti, in which the duct is absent in the
adult. In the former group are included the carp,
salmon, and eel; in the latter, the cod, bass, and perch* The basis for this distinction between the two groups, however, is far from invariable and many exceptions occur in both,” Ballantyne, (1927), set forth his ideas concern ing the probable evolutionary history of the gas blad der from the bilaterally symmetrical, ventrally placed, paired lungs to the dorsal and unpaired gas bladder of the modern fish. The transition from the air-bladder as It is seen in Amia, to the condition shown by the air-bladder of Salmo is primarily a still further re duction of the respiratory, and a fuller development of the hydrostatic function, with the accompanying changes in the form and blood supply of the organ, Ballantyne states:
"The evolutionary stages, as far as they can be traced, seem to be: first, normal, ventral, and paired lungs with the usual pulmonary blood and nerve supply: secondly, the gradual reduction of the left and enlargement of the right lung, seen in its early stages in Polypterua and completed in the adult
Ceratodus , This predominance of the right over the left lung Is possibly due in large measure to the position of the stomach well down to the left side.
The next stage Is illustrated by Amia with Its "pu'lmonoid" air-bladder, which has the normal pul monary nerve and blood supply, but opens dorsally Into the alimentary canal, Lepldosteous, again, has -12- a pulmonold air-bladder with pulmonary nerves, but blood coming from the aorta. In Acipenser the reduc tion of the respiratory function is complete and the air-bladder i3 a simple membranous sac with its blood supply from the dorsal aorta. In Salmo the hydrostatic function has developed further and the air-bladder has now "red-glands" In its anterior walls, but it is still a simple sac opening dorsally into the alimentary canal."
The earliest references to Physoclistous fishes were those of Rathke, (1827), and vonBaer, (l83lj.), who state that air-bladders develop as diverticula in open communication with the alimentary canal, the duct in later stages becoming more or less completely obliterated. Goodrich, (1930), states that physoclistous forms occur occasionally among physostomous groups, and open ducts are retained sometimes in physoclistous groups, therefore these terms have been abandoned as of little value In taxonomy. Goodrich also states: "Concerning the original position of the dorsal opening the evidence of embryology is ambiguous, as the bladder appears to arise sometimes on the left. These appearances are, however, probably deceptive, and due to differential growth and secondary torsions which are known to occur in the later stages or development*" Morgan, (1936), made a study of the fan-tail darter, Catonotus flabellarls flabellaris Rafinesque to determine whether this darter has a gas bladder at any time in its life history. Morgan studied embryos of this species in different stages of development and found in a 3~k rnm» embryo a diverticu lum of the foregut in the region of the second body
somite corresponding in position to its homologue in
species which as adults possess a gas bladder.
Morgan states, "Prom the structure of this diverticulum in Catonotus flabellaris flabellaris. and its relation to the foregut, body somites, and
the region of the heart, there is every reason to believe that this structure is a rudimentary air bladder which very soon completely disappears".
McEwen, (19^4-0) $ in his study of the development of the swim bladder in Hemlchromls blmaculata found that it arises as a dorso-lateral outgrowth from the gut. As to the circulatory elements of the swim bladder McEwen states the following, "It is to be noted that in the adult fish four oval areas of blood capillaries (the retia mirabilia) exist in the ventro lateral region of the bladder in its anterior half. -i4- From these areas blood vessels converge to form a vessel passing down through the secondary connection to join the artery from the dorsal aorta to the stomach pouch. 11
Hoar, (1937), states that "a part of the air bladder of the Atlantic salmon, Salmo aalar. origi nates as a solid mass of mesodermal cells dorso lateral to the gut. Later an epithelial evagination of the gut invades this mass and forms a tube lined by a syncytium whose cells are columnar at the pos terior end". Presumably such an air bladder has a lining of endodermal origin, surrounded by tissues in its walls of mesodermal derivation.
In a study of the origin of the gas bladder in the bluegill, Lepoml3 macrochirus by Duwe, (1952), the gut was found lying on the embryo’s left side and the gas bladder diverticulum was found evaginatlng from the right side of the gut toward the midline of the embryo. In this species the pneumatic duct and rete mirabile are transient structures, the duct be coming completely obliterated and the rete so reduced as to render its function doubtful. In the defini tive condition the gas bladder comes to lie dorsal to the gut. -15- A summary of the literature cited above indicates that fishes can be placed in four categories based on the structure and the presence or absence of the gas bladder* There are those which have two (carp) or three (suckers) chambered bladders and a duct of com munication with the gut which is designated the pneu matic duct. These gas bladders are referred to as of the physostomous type* A second group consists of those fishes which have a single chambered gas bladder which also is connected to the gut by a pneumatic duct. This type is also designated physostomous and is found in the pike, salmon, sturgeon and trout. A third type is designated as physoclistous and has a single chambered gas bladder without a pneumatic duct in the adult fish. Representatives with this type of gas bladder are the perch and the Centrarchid fishes, a family of fishes including the bluegill and green sunfish. A fourth category includes those fishes which have no gas bladder as adults. The darters generally are in this group. Pishes in this category could be referred to as aphysal as suggested by
Dr. J. W. Price. In the case of the more specialized Physoclistous type of gas bladder, as found for example in the blue gill, It is possible to trace, at least in part, a -16- phylogenetic recapitulation of the more primitive
physostomous type. At first, a diverticulum from
the gut gives rise to a pneumatic duct and a gas bladder. In later developmental stages specializa tions occur such as the atrophy of the pneumatic duct, the development of the gas gland and the
appearance of a rete mirabile. Before reaching
the definitive state, the rete undergoes a partial regression, rendering its function doubtful in the
adult condition.
As to their mode of origin, gas bladders arise
in one of three ways: 1. As a diverticulum from the
gut. 2. As a solid mass of mesodermal cells which
later is Invaded by an endodermal evagination from the gut. 3* As a solid mass of cells originating in the region of the esophagus and In which later appears a cavity communicating with the esophagus.
No direct correspondence appears to exist be
tween the modes of origin of the gas bladder in dif ferent fishes and the definitive condition In the
adult. It is known that either physostomous or
physoclistous fishes may have gas bladders originat ing as diverticula from the gut and these at various places on the gut, Type 1. It Is also known that In certain physostomous types the gas bladder arises as -17- a solid clump or cells, Types 2 or 3# It remains for the present writer to demonstrate a case of a physoclistous type of gas bladder arising as a solid mass of cells, a modification of Type 3 above. -18- MATERIALS AND METHODS Embryos and adults of Lepomis cyanellus were studied. Embryos of the blue gill and rainbow trout were examined as comparative material as were adult golden redhorse suckers.
The green sunfish embryos used In this study were reared by the writer in the laboratory. When males were observed to be nest-building In the waters of the Columbus, Ohio area, particularly In Mirror
Lake on the Ohio State University campus, collecting was started. It was a relatively simple matter to obtain the males because they were nest-building on gravel near the shore In water six to twelve Inches deep. The females were more difficult to obtain as they had not moved into the nests but remained in deeper water. On obtaining the females they were found to be gravid and the eggs were ripe even though the females had not moved to the nests.
Eggs were stripped Into petri dishes containing one fourth inch of distilled water. The milt was then stripped Into the dishes immediately and the contents swirled around with a fine rabbit-hair brush. It was found by trial and error that It is best to strip the eggs first because the short period -19- of fertilizability of the sperm results In fewer fertilized eggs if done conversely.
It was found that the fewer the number of embryos per dish the lower was the mortality rate. The author
usually kept thirty embryos in a dish. Water evapora
ted rapidly due to the large surface area and water was added at intervals to keep the level at one
fourth inch* Dead embryos were removed because the
mold which grew on them resulted In the entanglement
of living embryos and their subsequent death. Embryos were easily handled with rubber bulb pipettes, the tips being cut off to fit the embryos.
The embryos were incubated at room temperature.
The water temperature, measured at various intervals
day and night, fluctuated between the extremes 78° F and 83° F. Embryos were taken from the culture dishes at
Intervals varying from three to twelve hours and fixed In Bouin’s fixing reagent. At 292 hours fix
ation of embryos was stopped. At that time all yolk
had been absorbed and the embryos were becoming mal
formed and dying at a rapid rate. Embryos of the following numbers of hours of
incubation were studied; I4.7 , 5 0 , 5 3 , 5 7 .5 , 61*-.5 8 8 , 7 2 , 8 1 , 90, 1 0 0 , 10lj..5, 111*..5, 120.5, 127, 136, ll|4, -20- 1 5 2 , 1 6 0 , 1 6 8 , 1 7 6 , I8I4., 1 9 6 , 2 0 8 , 2 1 7 , 2 2 0 , 2 3 2 , 2 4 4 , 2 5 6 , 2 6 8 , 2 8 0 , and 2 9 2 .
These embryos were dehydrated by the alcohol series method and cleared In xylene. All stages, while in xylene, were placed in an imbedding oven for one hour. The xylene was replaced by imbedding paraffin which was changed after one hour. To insure thorough impregnation the embryos were left in the paraffin over night.
After imbedding the embryos in paraffin, sec tions were cut at eight microns. Sagittal sections were made of all stages, transverse sections of the critical stages only.
Paraffin was dissolved from the sections by xylene and the slides were taken down the alcohol series from 100% to 3 5 /£, were washed in water and placed in Mallory's phospho-tungstic acid hematoxylin. This stain was found by trial to be versatile and to give very good results.
As critical sections of each embryonic stage of development were located they were marked and photo micrographs of them were made. Gross dissections were made of the golden red- horse sucker in an attempt to locate the nervous and -21- vascular elements or the gas bladder.
Dissections were made of adult green sunfish and the nervous and vascular elements of the gas bladder were traced. A drawing was made of these
elements. The gas gland and rete mirabile of a green sunfish were excised and sectioned by the paraffin method at ten microns. After staining with hema
toxylin and eosin a microscopic examination was made and photomicrographs of typical sections were
taken.
In studying the origin and development of the
gas bladder and associated structures of the green
sunfish the older stages of development were sec
tioned first and sectioning of successively younger
embryos was continued until no trace of the gas bladder was found. -22- OBSERVATIONS Hatching occurred at thirty one hours of incu bation.
Embryos Incubated forty seven hours or fewer
showed no sign of the gas bladder anlage, (Figs, 1 & 2) At forty seven hours of development, the
notochord and nerve cord are In an advanced state
of development* The gut is well developed having at this stage a complete and open lumen.
The first manifestation of the gas bladder
appears in the fifty hour embryo, (Figs. 3 & if) At this stage the gut lies on the embryo’s left
side. The first appearance of the gas bladder
anlage Is seen as a solid clump of undifferentiated
cells lying adjacent to the gut on the midline
immediately under the notochord. This clump of
cells is observed at the level of the third body
somite. This solid cell clump Is compact and dis
tinct from the gut, and wholly cut off from the
lumen In the latter. However, by tracing backward
from later stages, this cell clump can be identified
as the anlage of the gas bladder which later becomes connected to the gut by a pnaumatic duct. Apparently the clump of cells or gas bladder anlage arises at -23- the time of gut formation but is not identifiable
as such until an increase in cell number takes place.
A sagittal section of a fifty three hour embryo
shows a progressive orientation of the cells in this solid cell mass and an increase in the size of the
gas bladder anlage as a whole. (Fig. 5) In the fifty seven and one half hour stage there is a further increase in size of the anlage
and an elongation of it. (Fig. 6 ) A transverse
section of this same stage shows an increase in the
size of the anlage and also strands of cells extend ing from this cell mass dorsad and ventrad to the gut. This condition probably results from an In
crease in cell numbers of the cell mass and their
epiboly toward the gut. (Fig. 7)
At sixty four and one half hours a sagittal
section shows that the clump of cells is no longer
elongate but has become rounded. (Fig. 8 )
Figure nine Indicates the condition at sixty
eight hours In which a further increase In the size of the anlage has taken place and the rounded condi tion is retained. A transverse section at this stage of development indicates further epibolic activity resulting in a connection between the outer wall of -2lp- the gut and the gas bladder anlage the latter lying immediately beneath the dorsal aorta* A small opening lies between the cell strands thus formed* When traced backward from later stages, these strands of cells are seen to be the anlage of the pneumatic duct. (Pig. 1 0 ) At seventy two hours the gas bladder anlage has a small lumen. (Fig. 1 1 ) Transverse sections of this stage of development show an elongation of the pneu matic duct anlage. (Fig. 12) In an adjacent section an outpocketing of the right side of the gut appears between the cell strands of the pneumatic duct anlage.
(Fig. 1 3 ) The lumen of the gut in this one section is seen to be elongated toward the midline of the embryo. A sagittal section of a seventy eight hour embryo shows the gas bladder to be connected by the pneu matic duct with the foregut. (Fig. 1I4.) A lumen is definitely present in the gas bladder. Transverse sections of this stage show the presence of a defin ite lumen in each of the three structures, the gas bladder, pneumatic duct and foregut, but without confluence, one with another. (Fig. 15) Figure sixteen indicates at eighty one hours a continuity between the lumena of the pneumatic -25- duct and of the gas bladder. At this incubation time the histology of the gas bladder and pneumatic duct are the same. A transverse section of this same stage of development, (Pig. 17), points up the continuity of the alimentary canal and the pneumatic duct. At this stage then the lumena of the gut, pneumatic duct, and gas bladder are continuous.
The continuity of the lumena of these three struc tures, present at eighty one hours of incubation is not seen in stages either preceding or following this one. The continuity of the lumen of the foregut with that of the pneumatic duct persists until the latter regresses: but the continuity between the lumena of the gas bladder and of the pneumatic duct, present at eighty one hours has apparently been lost by the next stage, ninety hours of incubation. At ninety hours the gut still lies on the left side of the embryo and the pneumatic duct proceeds from it to the median line. At this stage the gas bladder lies on the midline just ventrad of the dor sal aorta. A thickening of the anterior end of the gas bladder is a formative stage of the gas gland.
(Pigs. 18 & 19). The pneumatic duct and gas bladder cannot be seen together in any one transverse section due to the elongation of the duct and the caudad - 2 6 - growth or the bladder. (Pig. 20) Figure twenty one shows the junction of the gut and pneumatic duct in the one hundred hour embryo. The black spots above the gas bladder are pigmented epithelium and arise in this stage at about the same time as the pigmented epithelium of the eye.
Observations of the one hundred four and one half hour embryos show a thinning of the wall of the gas bladder, a thickening of the tissue of the ga3 gland, and a more definitive condition of the pneu matic duct, indicated by an increase in the size of the lumen and the thickness of its walls. The mid gut is thickened forming the stomach. The heart is in a highly developed condition. (Fig. 22) The major change from one hundred four and one half hours to one hundred twenty and one half hours is not in the pneumatic duct which remains open but in the size increase and regularity of the cells in the gas gland and in the growth and backward exten sion of the gas bladder. In this sixteen hour period the caudal margin of the gas bladder has developed caudally from the level of the sixth body somite to the level of the eighth body somite. (Figs. 23 & From one hundred twenty and one half hours to one hundred twenty seven hours the major change is In -27- the almost complete absorption of the yolk and the rapid increase in development of the liver and stomach. It should be noted that the gas gland at this stage is made up of columnar epithelium, (Pig, 25) As a result of the absorption of yolk there is a progressive rotation of the gut until the stomach and pneumatic duct at one hundred and thirty six hours come to lie on the dorso-ventro midline of the embryo as witnessed by their posi tion directly under the nerve cord, (Fig. 2 6 )
Figure twenty seven shows clearly the open pneumatic duct. The wall of the gas bladder Is as thin as It is In the definitive condition. The cells of the gas gland on close scrutiny can be seen to be In a state cf transition, from columnar to cuboldal. This one hundred forty four hour stage is the latest In which the supposedly ancestoral condition of an open pneumatic duct is seen. Figures twenty eight and twenty nine are taken from two different Individuals at one hundred fifty two hours of development. Individual differences as well as similarities in development can be seen. In either case there Is but a trace of a lumen remain ing in the pneumatic duct. In both cases the tissue - 28- of the gas gland is cuboidal in nature. These individuals however differ in body size, size of gas bladder, and location of the bladder. At one hundred sixty hours of development the rete mirablle. a network of capillaries supplying the gas gland, has been established. The cells of the gas gland are now of squamous structure, this condition being retained in the definitive gas gland. The lumen of the pneumatic duct has disap peared completely and all that is retained are some strands of tissue through which run the vascular elements supplying and draining the rete mirabile. The strands of tissue, joining the anterior end of the gas bladder to the posterior end of the esopha gus and through which the vascular elements course, will be referred to as the anterior ligament. (Fig. 30 The anterior ligament is not seen in this figure but will be seen in later ones) The condition of the gas bladder and associated structures remains rather constant from one hundred and sixty hours through one hundred and ninety six hours. (Figs. 30* 31* 32, 33, & 3l|J Any differences noted in these figures result from the representative Sections being taken at slightly different levels. -29- The anterior ligament is visible in figure thirty four because this section is laterad to the midline of the individual.
At two hundred and eight hours of development the gas bladder artery can be seen coursing from the dorsal aorta to the rete mirabile. The gas bladder vein is visible and will be seen in later stages to empty into the hepatic portal vein.
This artery and vein constitute the primary vascular elements in relation to the adult gas bladder. (Pig. 35)
From two hundred and eight hours through two hundred and ninety two hours little change is seen in the development of the gas bladder, gas gland, and rete mirabile. Figure forty two shows the hepa tic portal vein in the two hundred sixty eight hour individual. It should be noted here that body develop ment after two hundred sixty eight hours becomes anomolous probably as a result of starvation inas much as the yolk has been completely utilized and no food was introduced into the water. (Figs. 35-ifij-)
The adult condition as seen in figure forty five, agrees with the condition as found at two hundred and eight hours of development. The rete mirabile in the adult is visible to the naked eye as a red mass overlying the oval shaped, opaque gas gland. The gas bladder artery and vein supplying
and draining the rete mirabile are clearly visible within the anterior ligament but the capillaries
of the network of which the rete is composed cannot
be seen with the unaided eye. The nerve elements
of the gas gland were not visible in the develop mental stages probably because the techniques in
volved were not specific for nerve tissue although
these elements are easily seen in the adult. The
gas bladder itself is a delicate single-layered mem branous sac closely overlaid by peritoneum. The
tissue of the anterior ligament surrounds and is
interspersed between the vascular and nerve elements
supplying the gas bladder.
A microscopic examination of the rete mirabile
shows a dense network of capillaries branching and anastomosing in such a manner that it is difficult
to locate one cell of the gas gland that does not lie adjacent to a capillary. The larger circulatory
elements appear to be masses of capillaries coursing parallel to each other. (Pig. 14-6 ) -31- The narv© elements of the gas bladder lie in the rete mirabile and consist of clumps of ganglion cells and their processes radiating out between the capil laries, (Pigs, I4.7 & L|.8 ) -32-
d i s c u s s i o n
From the preceding section it is established that the green sunfish develops a physoclistous type of gas bladder which between seventy eight hours and one hundred forty four hours of development is of the ancestral physostomous type. (See diagrams, page 37) The first indication of a developing gas bladder occurs at fifty hours as a clump of undifferentiated cells lying to the right of the gut which is displaced to the left of the midline of the embryo. (Diagrams 1 and 2) This mode of origin is in sharp contrast to that usually ascribed to both the physostomous and to the physoclistous fishes, wherein the gas bladder is generally regarded as originating as a diverticulum of the foregut. For example, the present writer has observed this latter mode of origin in the rainbow trout, a physostomous fish, and also in the bluegill, a physoclistous fish. Inasmuch as the bluegill,
Lepomls m. macrochirus. and the green sunfish, Lepomls cyanellus, are members of the same genus, it is of considerable interest that these two closely related species develop their gas bladders in distinctly dif ferent ways. However, similar differences in the -33- manner in which corresponding structures develop in different fishes of the same genus have been reported in the genus Coregonus (Vogt, I8I4.2 ) and in the genus Salmo (Hoar, 1937)* Hence, the findings reported in this paper are not inconsistent with conditions reported elsewhere nor wholly unexpected.
With the clump of undifferentiated cells, the anlage of the gas bladder, established at fifty hours, the pneumatic duct anlage next develops, between sixty eight and seventy eight hours of incu bation, In its first manifestation the duct con sists of cells growing out from the cell mass of the gas bladder anlage in such a way as to form cell strands which connect with the ventral and dorsal margins of the foregut. (Diagram 3) Between the points of junction of the cell strands and the gut a small outpocketing of the gut occurs, (Diagram Jp) This outpocketing probably is a result of mutual interaction between the cells of the pneumatic duct anlage and the cells of the gut. This outpocketing of the gut does not persist beyond the stage when it appears. By the next stage the gut wall at this point has perforated, and thus has brought about the -3 k-~ confluence or the lumena of the gut and the pneu matic duct. Further alignment of the cells of the gas bladder, at the point of origin of the cell strands of the pneumatic duct anlage, results in continuity of the lumena of the gut, pneumatic duct, and gas bladder. (Diagram £) The continuity of the lumena of these three structures does not persist however and by the next stage the pneumatic duct becomes separated from the gas bladder but retains its con tinuity with the gut. (Diagram 6) The pneumatic duct retains its open lumen from seventy eight hours through one hundred fifty two hours of development after which time it closes completely, (Diagram 7), and is retained thereafter only as the anterior liga ment extending from the gas bladder to the esophagus. (Diagram 8) In the definitive condition, the artery, vein, and nerve of the gas bladder are found in this anterior ligament. (See fig. 1^5, p. 88) The gas gland In its development at the anterior end of the gas bladder is first composed of columnar cells. (Diagram 6) Later, Its cells become cuboidal,
(Diagram 7), and still later squamous, (Diagram 8), the latter condition being definitive. -35- The rete mirabile consists of a dense network of branching and anastomosing capillaries coursing between the cells of the gas gland. A branch of the vagus nerve runs to the gas gland. Clumps of ganglion cells can be seen in the rete mirabile. and nerve twigs branch out between the capillaries. (Pig. l\.6, p. 92 and Pig. P* 9l|) On the basis of structure it is deemed probable that the rete mirabile and the gas gland function in gas production and absorption. However, the physiology of these structures has not been studied by this author. It is reasonable to assume that these structures function in a manner similar to that found in related species by numerous investi gators in the field of fish physiology. - 36 - A Schematic Review of Development of the Gas Bladder Diagram 1, The undifferentiated cell clump, gas bladder anlage, appears to lie dorsal to the foregut but not in contact with it in this sagittal section* Diagram 2. The gas bladder anlage in reality lies medio-
dorsad to the foregut and directly under the notochord as seen in this transverse section of early development. Diagram 3» This transverse section shows a small lumen in the gas bladder anlage. Cell strands, the pneumatic
duct anlage, grow out from the gas bladder anlage and
form a junction with a slight out-pocketing of the gut. Diagram It. A definite lumen in gas bladder and pneu
matic duct. No continuity of lumen of gut, pneumatic duct, and gas bladder. Diagram 5. Histologically the gut, pneumatic duct, and
gas bladder are similar. Lumena of gut, pneumatic duct, and gas bladder are continuous.
Diagram 6. Gas bladder lies above stomach, is thin walled
and is no longer continuous with the pneumatic duct. The gas gland is present and consists of columnar cells.
Diagram 7. Increase in gas bladder size, gas gland of cuboidal cells. Lumen of pneumatic duct closing. Diagram 8. Further increase in gas bladder size, gas gland has definitive squamous cells. Pneumatic duct retained only as the anterior ligament. minimum
9 m pillllUaam,,,,, mmuimixmrnTnwannnmi
TTTTfmm iii mi in m um HT'rrmT ^ iimmnin mil in miinmiijg 0 8
z 1 -iC- -38-
s u m m a r y 1. A study was made of the origin and development of the gas bladder and associated structures in the green sunfish* 2* The first manifestation of the gas bladder itself appears in the fifty hour stage of develop ment at room temperature as a clump of undifferentiated cells mediad to the gut which is displaced to the left
of the embryo. 3. The pneumatic duct anlage as a second element first appears as solid cell strands originating from the gas bladder anlage and connecting the gas bladder
anlage to the gut at fifty seven and one half hours*
If. A continuous lumen in the gas bladder and pneu matic duct and gut is seen only at eighty one hours.
Thereafter the distal end of the pneumatic duct is occluded. 5* At ninety hours the gas gland makes its appear
ance as a thickened mass of cells at the anterior
end of the gas bladder. The gas gland in development goes through stages in which it has columnar, cuboidal and squamous cells successively. The latter is the definitive condition* -39- 6. At one hundred fifty two hours of development the lumen of the pneumatic duct is obliterated* The por
tion of the duct which is retained is referred to as the anterior ligament. This ligament is made up of
tissue strands. Later through it course the vascular and nerve elements of the gas bladder.
7. At one hundred sixty hours the rete mirabile, a
network of capillaries supplying the gas gland, has
been established.
8. At two hundred and eight hours of development the gas bladder artery and gas bladder vein have been fully established. The gas bladder artery is a branch
of the coeliaco-mesenteric artery. The gas bladder
vein empties into the hepatic portal vein. 9. The two hundred eight hour individual and all subsequent stages are equipped, as is the adult, with a gas bladder, gas gland, gas bladder artery, gas
bladder vein, and anterior ligament. 10. In the adult condition a branch of the vagus nerve
courses through the anterior ligament, terminating in
the rete mirabile and gas gland. This branch is refer
red to as the gas bladder nerve. -to ll* The adult rete mirabile is an extensive network of branching and anastomosing capillaries closely- overlying and interspersed within the tissue of the gas gland. It contains clumps of ganglion cells and nerve twigs dispersed among the capillaries* 12. The gas bladder of the adult is a thin membranous sac consisting of a single layer of squamous cells, lying dorsal to the other viscera, ventral to the dorsal aorta, and bounded at Its anterior and pos terior ends by the extremities of the body cavity*
13. The green sunfish thus becomes equipped with a physoclistous type of gas bladder which, during its development, recapitulates in part the physostomous type of gas bladder, but which originates, not as a diverticulum of the foregut, but as an Independent
3olid mass of undifferentiated cells. llj.. To the author* s knowledge, this Is the first demonstrated instance of a physoclistous type of gas bladder originating In the manner described above. -1+1 - ABBREVIATIONS USED ON FIGURES A ------Auricle AC ------Alimentary canal AL ------Anterior ligament AUD ------Auditory vesicle
A W ------Auriculo-ventricular valve B ______Brain
BA ______Gas bladder artery
CGG ------Cell of gas gland
CRM ------Capillary of rete mirabile DA ------Dorsal aorta
E Esophagus
FG Foregut G ______Gall bladder GA ______Gill arch
GB ______Gas Bladder
GBA Gas bladder anlage GBV Gas bladder vein GC Ganglion cells GE ______Gut evagination GG - Gas gland GV ______Gastric vein H Heart - 1* 2- HG ------Hind gut HPV ------Hepatic portal vein
K Glomerulus of mesonephric kidney L ------Liver M ------Myotome
MG ------Midgut N ------Nerve cord
NC ------Notochord
NT ------Nerve twig
P ------Pronephric tubule
FD ------Pneumatic duct
PDA ------Pneumatic duct anlage
RM ------Rete mirabile S ------Stomach
SV Sinus venosus V ______Ventricle
VRM ------Blood vessel of rete mirabile
Y ------Yolk -to-
Figure 1 Photomicrograph of a sagittal section of a green sunfish at I4.7 hours of development.
Magnification 150X.
Figure 2. Photomicrograph of a transverse section of a green sunfish at I4.7 hours of development.
Magnif ication 1±?0X. Pig. 2 - 1* 5-
Figure 3 Photomicrograph of a sagittal section of a green sunfish at 50 hours of development«
Magnification 15>0X,
Figure I4. Photomicrograph of a transverse section
of a green sunfish at $ 0 hours of development.
Magnification 15>0X. Pig. 3
Pig. k -4 7 -
Figure 5 Photomicrograph of a sagittal section of a green sunfish at 53 hours of development.
Magnification 150X.
Figure 6 Photomicrograph of a sagittal section of a green sunfish at 57*5 hours of development•
Magnification 150X. -48 -
Fig. 5
Fig. 6 Figure 7
Photomicrograph of a transverse section of a green sunfish at 57*5 hours of development.
Magnification 150X.
Figure 8
Photomicrograph of a sagittal section of a green sunfish at 61j.,5 hours of development.
Magnification 150X,
-51-
Figure 9 Photomicrograph of a sagittal section of a green sunfish at 68 hours of development. Magnification 150 X.
Figure 10 Photomicrograph of -a transverse section of a green sunfish at 68 hours of development. Magnification 150 X. PDA
Fig. 10 -53-
Figure 11 Photomicrograph of a sagittal section of a green sunfish at 72 hours of development#
Magnification 150 X.
Figure 12 Photomicrograph of a transverse section of a green sunfish at 72 hours of development*
Magnification 150 X# Pig. 12 -55-
Figure 13 Photomicrograph of a transverse section of a green sunfish at 72 hours of development• Magnification 150 X.
Figure 1I4. Photomicrograph of a sagittal section of a green sunfish at 78 hours of development.
Magnification 150 X. Fig. llj. Figure If? Photomicrograph of a transverse section of a green sunfish at 78 hours of development.
Magnification 150 X,
Figure 16 Photomicrograph of a sagittal section
of a green sunfish at 81 hours of
development.
Magnification 150 X. Fig. l6 -59-
Figure 17 Photomicrograph of a transverse section of a green sunfish at 8 1 hours of development. Magnification 150 X,
Figure 18 Photomicrograph of a sagittal section of a green sunfish at 9 0 hours of development. Magnification 150 X, Pi2. 18 - 61 -
Figure 19 Photomicrograph of a sagittal section of a green sunfish at 9 0 hours of development. Magnification 1$0 X,
Figure 20 Photomicrograph of a transverse section of a green sunfish at 9 0 hours of development. Magnification 150 X, i®sn
Fig. 19 -63
Figure 21 Photomicrograph of a sagittal section of a green sunfish at 1 0 0 hours of development. Magnification 1^0 X*
Figure 22 Photomicrograph of a sagittal section of a green sunfish at lQlj.,5 hours of development* Magnification l£0 X. Fig. 22 - 6 5 -
Figure 23 Photomicrograph of a sagittal section of a green sunfish at lli^.,5 hours of development. Magnification 150 X.
Figure 2I4. Photomicrograph of a sagittal section of a green sunfish at 1 2 0 . 5 hours of development.
Magnification 150 X. Fig. 2k -67-
Figure 25 Photomicrograph of a sagittal section of a green sunfish at 1 2 7 hours of development. Magnification 150 X.
Figure 26 Photomicrograph of a sagittal section of a green sunfish at 1 3 6 hours of development. Magnification 150 X. Pig. 26 -69-
Figure 27 Photomicrograph of a sagittal section of a green sunfish at lijij. hours of development. Magnification l£0 X,
Figure 28 Photomicrograph of a sagittal section of a green sunfish at 1 $ 2 hours of development. Magnification 150 X, Pig. 28 0
-71-
Pigur© 2 9 Photomicrograph of a sagittal section
of a green sunfish at 1 $ 2 hours of development. Magnification 1^0 X.
Figure 30 Photomicrograph of a sagittal section of a green sunfish at IjSO hours of development•
Magnification 150 X. ■*•4.
Fig. 29
4.
Fig. 30 -73-
Figure 31 Photomicrograph of a sagittal section of a green sunfish at 1 6 8 hours of development. Magnification 150 X.
Figure 32 Photomicrograph of a sagittal section of a green sunfish at 1 7 6 hours of
development.
Magnification 150 X, - 1 k -
Fig. 32 -75-
Figure 33 Photomicrograph of a sagittal section of a green sunfiah at I8I4. hours of development* Magnification 150 X,
Figure 3lf Photomicrograph of a sagittal section of a green aunfiah at 196 hours of development. Magnification 150 X, Pig. A -77-
Figure 35
Photomicrograph of a sagittal section of a green sunfish at 208 hours of development*
Magnification 150 X.
Figure 36
Photomicrograph of a sagittal section of a green sunfish at 217 hours of development.
Magnification 150 X. i
Pis- 35
Pig. 36 -79-
Figure 37 Photomicrograph of a sagittal section of a green sunfish at 220 hours of development*
Magnification l£0 x«
Figure 38
Photomicrograph of a sagittal section of a green sunfish at 232 hours of development*
Magnification 150 X* Pig. 37
Pig. 38 -81-
Figure 39
Photomicrograph of a sagittal section of a green sunfish at P)\)\. hours of development.
Magnification 150 X.
Figure lj.0
Photomicrograph of a sagittal section of a green sunfish at 256 hours of development.
Magnification 150 X. issisii
Pig. 39
Fig. 1|0 -83-
Figure Ij.1 Photomicrograph of a sagittal section of a green sunfish at 268 hours of
development.
Magnification 15>0 X.
Figure i\.Z
Photomicrograph of a sagittal section
of a green sunfish at 268 hours of development.
Magnification 1$Q X. Pig. 41
Pig. 1+2 -85-
Figure Ij-3
Photomicrograph of a sagittal section of a green sunfish at 280 hours of development. Magnification 150 X*
Figure
Photomicrograph of a sagittal section of a green sunfish at 292 hours of development*
Magnification 150 X. Pig. Ljl}. -87-
Figure
Diagram of the gas bladder and associ ated structures of an adult green
sunfish.
Magnification X, I CO CDI
G -(\st,ri c. V tin
Gas 0Ic<. Pig. b 5 - 89- Figure lj.6 Photomicrograph of a frontal section of the gas gland and rete mirablle of an adult green sunfish# Magnification 150 X# -90- Flg. i)-6 -91- Figure 4.7 Photomicrograph of a frontal section of the gas gland and rete mlrablle of an adult green sunfish# Magnification 1 $ 0 X# Fig. k 7 -93- Figure I4.8 Photomicrograph of a frontal section of the gas gland and rete mlrabile of an adult green sunfish. Magnification 150 X. Fig, 14.8 -95- bibliography 1. Agassiz, J. L* R. Remarks on the development of the air bladder In fishes. Proc. Amer. Assoc. Adv. Sci., 1850, Vol. 3*, P. 72. 2. Ballantyne, P. M. Air-Bladder and lungs. Tr* Royal Soc. Edinburgh., 1926-27, Vol. 55., Parts 1 & 2., Pp. 371-393. 3. Duwe, A. E. The Embryonic Origin of the Gas Bladder In the Gentrarchid Pish Lepomls macrochlrus macrochirua♦ Copeia 1952 No. 2 P. 92 if. Evans, H. M. Anatomy and physiology of the air-bladder and Weberian ossicles in Cyprini- dae. Proc. Royal Soc. London., 192L}.-25., Series B., Vol. 97*, Pp. 5^5-576. 5. Goodrich, E. S. Studies on the structure and development of the vertebrates. Macmillan Co., 1 9 3 0 ., Pp. 578-583. 6 . Hall, F. G. The functions of the swimbladder of fishes. Biol. Bull. 19214-., Vol. 14-7., No. 2., Pp. 79-117. 7. Hoar, W. S. Development of the Swimbladder of the Atlantic Salmon. J. Morph., 1937, Vol. 6 l, Pp. 309-319. -96- 8 * Houghton, W. The air or swimming bladder of fishes. Pop. Sci. Review. 1868., Vol. VII., Pp. 378-385. 9# Kerr, J. E. Textbook of Embryology. Vol. II. Vertebrate with the exception of mammalia. Pp. 173-17l|-. Macmillan and Co. London, 1919* 10. McEwen, R. S. The Early Development of the Swim bladder and Certain Adjacent Parts In Hemichromis bimaculata. J* Morph., 1 9 J4-O, Vol. 6 7 , No. 1, July 1. 11. Morgan, G. D. A study of the vestigial air bladder in the darter, (Catonotis flabellaris, Rafinesque). PhD dissertation. The Ohio State University. 1936. 12. Morris, Charles. On the air bladder of fishes.Proc. Phil. Acad, of Nat. Sci. 1885* Pp. 12^-135. 13. Potter, G. E. The srwim-bladder of a 65 mm. Gar. pike (Lepidosteus platystomus) embryo* Proc. Iowa Acad. Sci. Vol. XXXII. 1 9 2 5 . Pp. I4.O7-J4.ll4.. 1I4.. Price, J. W. Embryology of the White Fish Coregonus clupeaformis. Ohio Jr. of Sci., 1935, vol. 35, no. 1 , Jan. -97- 15* Quekett, John, On a peculiar arrangement of blood vessels in the air-bladder of fishes* With some remarks on the evidence which they afford of the true function of that organ. Trans. Micr. Soc. London. Vol. I. 1 8)|J|. Pp. 99-108. 1 6 . Strieker, P. Plattenmodelle zur Entwichlung von Darm, Leber, Pankreas, und Schwimmblase der Porelle. Intern. Monatsschr. f. Anat. und Phys., 1899, Bd. l6, S. 1-26. 17* Tracy, H. C. The morphology of the swim- bladder In teleosts. Anatomischer Anzeiger. Vol. 38. 1911. Bp. 6 3 8 -64.8 . 18. Vogt, C. Embryologie des Salmones, (In Agassiz, L. Histoire Naturelle des Poissons D'eaus Douce De L'Europe Centrale), 1 8 4 2 , Neuchatel• 19* Wickliff, E. L. The internal anatomy of a fish and the function of each part. Bull. #5, State of Ohio. Dept, of Agri• DIv. of Conservation. Bureau of Scientific Research. Publication 1 9 4 ^ # -98- 20« Woodland, W. W. F. On the structure and function of gas glands and retia mlrabile associated with the gas bladder of some teleostean fishes. Proc. Zool. Soc. London. Vol. I. 1911. Pp. l83-2lj.8. AUTOBIOGRAPHY I, Arthur Edward Duwe, was born In Saginaw, Michigan, July 17, 1922* I received my secondary school education in the public schools of Saginaw graduating from Arthur Hill High School in 19il0o My undergraduate training was obtained at Alma College, Alma, Michigan, from which I received the Bachelor of Science degree in 19^4-9® While enrolled at Alma College I was an undergraduate laboratory assistant for one year and a half* I entered graduate school at the Ohio State Univer sity in 19^4-9 and received the Masters Degree in Science in 1950* The past three years have been utilized in completing the requirements for the degree Doctor of Philosophy. During my four years in The Graduate School of The Ohio State University I acted at various times In the capacity of graduate assistant, assistant instruc tor, and laboratory technician*