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

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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.

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

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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.