VETERINARSKI ARHIV 69 (2),
63-68, 1999

ISSN 1331-8055 Published in
Croatia

Response of aerial respiratory
organs of the
air-breathing catfish Heteropneustes fossilis
(Bloch)
to extreme stress of desiccation

Ram Sanehi Parashar and Tarun
Kumar Banerjee*

Histochemistry and Histopathology
Laboratory, Department of Zoology,
Banaras Hindu University, Varanasi, India

* Contact address:
Dr. Tarun Kumar
Banerjee,
Histochemistry and Histopathology Laboratory, Department of
Zoology, Banaras Hindu University, Varanasi-221 005, India,
Phone: 91-542
318 372; Fax: 91 542 317 074; E-mail: [email-masked]

PARASHAR, R. S., T. K. BANERJEE:
Response of aerial respiratory organs of the air-breathing catfish
Heteropneustes fossilis (Bloch) to extreme stress of desiccation. Vet.
arhiv 69, 63-68, 1999.

ABSTRACT

Histopathological alterations caused by
desiccation stress in the aerial (accessory) respiratory organs (ARO) of
Heteropneustes fossilis which has a developed bimodal respiratory
mechanism for exploitation of water (through its gills) as well as air (through
its ARO) have been described. The ARO assist the fish in surviving extreme
drought conditions. When out of water, even though the fish survive for about 16
h, their air sacs suffer extensive damage. In the initial stages the fish very
frequently open their mouths to gulp in more air. The blood channels of
secondary lamellae of the ARO, engorged with ridged blood channels, exhibit
extensive protrusion into the lumen where they form a network of very
thin-walled tube-like structures. Prolonged desiccation causes wear and tear to
these greatly extended blood channels, leading to haemorrhaging into the lumen.
Simultaneously, from the blind ends of the ARO, many of the ridges approach very
close to each other and finally meet, leaving no free respiratory surface in the
lumen to breathe aerially, ultimately resulting in failure of aerial respiration
and the demise of the fish.

Key words: accessory respiratory
organs, desiccation, Heteropneustes fossilis, histopatho-logy,
air-breathing catfish, India

Introduction

Heteropneustes fossilis, the
tropical air-breathing fish of the Indian subcontinent, inhabits derelict water
bodies having poor O2 conditions. These fish have a developed bimodal
respiratory mechanism for exploitation of water (via its four pairs of
teleostean gills) as well as air (via air-breathing organs, ARO). Hence, they
are able to withstand extreme drought conditions, especially when ponds become
dried up during the long summer period (MUNSHI, 1962, 1963). Also, it has been a
common practice to transport these fish in bamboo baskets, where they remain
alive even when they are out of the water for several hours. ARO in this fish
are a pair of sac-like structures which lie deeply embedded in the body myotomes
at the trunk region of both sides of the vertebral column. The ARO are adapted
to perform aerial respiration only and never come into direct contact with the
aquatic environment.

Therefore, efforts were made to
discover the histophysiological alterations taking place in the ARO of the
air-breathing catfish Heteropneustes fossilis prior to their death, while
combating desiccation stress and prevention of branchial respiration by
performing aerial respiration only when the fish are subjected to extreme stress
of desiccation. The efficacy of ARO in maintaining life has also been observed.

Materials and methods

Healthy specimens of Heteropneustes
fossilis (35-40 g body mass, 18-20 cm in length) were collected from a
single population at Varanasi and were acclimated for one month in tap water
(having dissolved O2, 6 mg/l, pH 7.5, water hardness 23.2 mg/l and
water temperature 24±2 °C) in large plastic aquaria in a laboratory. They were
fed with minced goat liver on alternate days and the water was renewed every 24
h, leaving no faecal matter, unconsumed food or dead fish, if any. Feeding
ceased 24 h prior to the commencement of the experiment, with starvation
continuing throughout the period of the experiment.

Four groups of ten acclimated fish
(15±1 cm in length) were removed from water and placed on dry plastic aquaria in
the laboratory for exposure to the air (atmospheric humidity 69%, room
temperature 27 to 30 oC) for 16 h, beyond which they rarely survived.
Control groups of fish were retained in tap water.

After the expiry of 0, 6, 12 and 16 h
of desiccation, five fish each from the experimental aquaria, as well as from
the control aquaria, were sacrificed. The entire branchial diverticulum (air
sac) from both sides of the fish was fixed in 10% neutral formalin and aqueous
Bouin's fluid. Six µm paraffin sections were stained with Ehrlich's or Harris
haematoxylin and alcoholic eosin (H/E) for routine histopathological analysis.
Certain carbohydrate moieties were visualised by periodic acid-Schiff (PAS)
after McManus, 1946, Alcian blue pH 2.5 (AB 2.5), AB 2.5/PAS, Alcian blue pH 1.0
(AB 1.0) and salivary amylase/PAS (PEARSE, 1985) techniques.

Results and Discussion

Control accessory respiratory organ

The inner respiratory surface of the
ARO, which is thrown into several folding or papilla-like ridges, is lined at
its inner surface with stratified epithelium (Fig. 1) and consists of vascular
and non-vascular areas. The vascular areas are characterised by the presence of
small and large respiratory islets (MUNSHI 1962, 1980, 1993). An islet (primary
lamella) is formed by double rows of secondary lamellae (SL), which hang freely
into the lumen. In the transverse section, the SL penetrate perpendiculary into
the stratified epithelium. The SL is composed of alternately arranged blood
channels (BC) and pillar cells (PC). Within the blood channels, one or two blood
cells are generally noticed. The single-layered respiratory epithelium separates
the blood in the SL from the air in the lumen of ARO. Mucous cells (MCs)
(secreting non-sulphated acidic mucin) and epithelial cells (ECs) constitute the
non-vascular areas. A thin layer of slime sometimes covers the epithelial
surface.

Next to the epithelium, and serially
arranged, are basement membrane, connective tissue, a thin membrane and muscular
coat. The fish regularly raises its snout above the surface of the water to
engulf air for aerial respiration and takes 40% of O2 through its air
sacs in saturated waters (bearing 3% O2) (MUNSHI and CHOUDHARY,
1994). Desiccation stress prevents normal branchial respiration. Hence, gasping
for air in these fish increased during the initial stages of the experiment.

Fig. 1. Transverse section of the air
sac of control H. fossilis,
showing its structural organisation. Note
the enormous space in the lumen (L). (PAS; ×35)
Fig. 2. Collapse of the air
sac following 6 h of desiccation. Note the gluing of the respiratory epithelia
of two approaching ridges. (HE; ×35)
Fig 3. Air sac of 16 h desiccated fish,
showing disappearance of air space in the lumen (L) due to adhesion of
epithelial linings of closely approximated neighbouring ridges. Note the strong
reaction in the mucous cells and the slimy secretion for sulphated
mucopolysaccharides. (Alcian blue/PAS reaction; ×200)

E=epithelium;
F=furrows, L=lumen; MC=mucous cells; P=pigment cells; R=ridges; S=slime.

Desiccated accessory respiratory
organ

Following desiccation the structure of
the air sacs of the desiccated fish showed massive damage. The immediate
response to the ARO is manifested by an enormous increase in density of RBC in
the channels of the SL, which become greatly extended and distend into the
lumen. Thus, the blood channels of the SL protrude into the lumen as
balloon-like projections. Subsequently, these channels form a network of very
thin-walled, blind tube-like structures over the epithelial surface. More
engorging of the blood channels continues as desiccation progresses. The gravity
of these thin-walled, tubular structures thus increases, causing the bending of
the blood channels of the SL, bending at 90° to lie flat on the inner surface of
the air sac. The network of thin-walled vascular channels also spread over the
non-vascular areas. The extensive network of the existing fine blood vessels in
the connective tissue immediately below the respiratory epithelium
simultaneously becomes engorged with RBC. All these manifestations substantially
decrease the barrier distance between the air in the lumen and the blood in the
sac-like SL. It is thought possible that this may increase the efficacy of the
air sac, albeit temporarily, by compensating for the failure of brachial
respiration. JOHANSEN et al. (1970) reported that when gill-breathing in a
primitive air-breather, Amia calva varies reciprocally with O2
tension of the air-breathing organ because blood bypasses the gas exchange blood
vessels of the gills where it is not the primary site for O2 uptake.
This is also true of H. fossilis, which could survive for several hours
even though the blood channels in the gills (PARASHAR and BANERJEE, 1998) also
show extensive engorgement with RBCs, mostly in the initial stages of
desiccation. Prolongation of dehydration beyond 16 h due to prolonged
desiccation causes wear and tear of these thin-walled extensively stretched
blood channels of the SL of the gills of H. fossilis, leading to severe
haemorrhaging and resulting in failure of branchial respiration (PARASHAR and
BANERJEE, 1998).

From the blind end of the air sacs,
many of the ridges come very close to each other and ultimately meet. This
blocks the free surface of the respiratory epithelium, causing a considerable
reduction in the volume of the lumen of the air sac and leaving very little
space for aerial respiration (Fig. 2). Often, a slightly eosinophilic, weakly
PAS positive viscous material of varying thickness glues the inner surfaces of
the two approaching ridges and chokes the lumen (Fig. 3). The MCs of the ARO of
the desiccated fish alter their staining property to secrete sulphated mucin and
keep the surface of the ARO moist for longer periods. However, all these
attempts to keep the ARO moist in order to maintain aerial respiration does not
last long, and the fish dies, ultimately due to haemorrhaging, dehydration of
vital organ systems and the collapse of many vital physiological processes (e.g.
CO2 and N2 elimination) resulting in total asphyxiation
due to failure of (aerial) respiration. Regular sprinkling of water increases
the survival period of the desiccating fish due to renewal of the water
molecules, which helps to maintain respiratory, as well as secretory processes
through the skin and gills (PARASHAR and BANERJEE, 1998). The other important
cause of death, which perhaps hastens the death, of this air-breathing fish is
the retention of toxic metabolic substances, such as CO2 and
N2, which in fish/amphibians are generally eliminated via the
skin/gills (HUGHES, 1966) because H. fossilis releases very little
CO2 into the air through its air-sacs (R.Q. being only 0.17) when it
respires in water (MUNSHI, 1993). Further, the CO2 tolerance limit of
H. fossilis is high in comparison to other (mostly pure water-breathing)
fish. Thus, the gas exchange continues for a considerable length of time, even
when the CO2 level in the water body of this fish is high (14.5% by
volume of air, MUNSHI, 1993), and the fish survive for longer periods.

Acknowledgements
This work was
supported by a grant-in-aid from the University Grants Commission, Government of
India, New Delhi. Research Project No. F 3-55/93 (SR-II)

References

HUGHES, G. M. (1966): Evolution between
air and water. In: Ciba Foundation symposium on Development of the lung (A. V.
S. de Reuck and R. Porter, Eds.). Churchill Ltd., London. pp. 64-80.

JOHANSEN, K., D. HANSON, C. LENFANT
(1970): Respiration in a primitive air-breather, Amia calva. Res.
Physiol. 9, 162-174.

MUNSHI, J. S. D. (1962): On the
accessory respiratory organs of Heteropneustes fossilis Bloch. Proc Royal
Soc Edinberg. 68, 128-146.

MUNSHI, J. S. D. (1980): The structure
and function of the respiratory organs of air-breathing fishes of India
Sectional (Zool. Entomol. and Fisheries) Presidential Address, Proc
67th Session, Indian Sci. Association. Part II. pp. 1-70.

MUNSHI, J. S. D. (1993): Structure and
function of the air-breathing organs of Heteropneustes fossilis. In:
Advances in Fish Research I (Singh, B. R., Ed.). Narendra Publishing House.
Delhi India. pp. 99-138.

MUNSHI, J. S. D., S. CHOUDHARY (1994):
Ecology of Heteropneustes fossilis (Bloch): an air-breathing catfish of
South East Asia. Freshwater Biological Association of India Bhagalpur, India.

PARASHAR, R. S., T. K. BANERJEE (1998):
Response of gills of the air-breathing catfish Heteropneustes fossilis to
desiccation stress. (in press)

PEARSE, A. G. E. (1985):
Histochemistry-Theoretical and applied. Vol. II. Edinburgh, London. Churchill
Livingstone Inc. pp. 441-1055.

Received: 4 May 1998
Accepted: 30
March 1999

PARASHAR, R. S., T. K. BANERJEE:
Odgovor zracnih disnih organa ribe dvodihalice Heteropneustes fossilis
(Bloch) na ekstremni stres isusivanja. Vet. arhiv 69, 63-68, 1999.

SAZETAK

Opisane su histopatoloske promjene,
izazvane stresom isusivanja, zracnih (pomocnih) disnih organa u dvodihalice
Heteropneustes fossilis koja je razvila dvostruki disni mehanizam, i to
iz vode (putem skrga) i iz zraka (putem zracnih disnih organa). Ribe s zracnim
disnim organima prezivljavaju ekstremne susne uvjete. Kada su izvan vode
prezivljavaju oko 16 sati i tada njihove zracne vrecice trpe opsezna ostecenja.
U pocetnoj fazi, riba cesto otvara usta kako bi uzela vise zraka. Zracni kanali
sekundarnih listica zracnih disnih organa obuhvaceni s grebenastim krvnim
kanalima pokazuju znatnu protruziju u lumen gdje tvore mrezu cjevolikih
struktura vrlo tankih stijenki. Produljeno isusivanje uzrokuje trosenje i
kidanje tih jako izduzenih krvnih kanalica i posljedicno krvarenje u lumen.
Istovremeno u slijepim krajevima zracnih disnih organa mnogi se grebeni
medusobno priblizavaju i spajaju, te tako vise ne ostavljaju slobodnu zracnu
disnu povrsinu s konacnim zakazivanjem zracnog disanja i ugibanjem ribe.

Kljucne rijeci: pomocni disni
organi, isusivanje, Heteropneustes fossilis, histopatologija,
dvodihalica, Indija

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