Ajay Kumar Mittal

Comparative analysis of innate immune parameters of the skin mucous secretions from certain freshwater teleosts, inhabiting different ecological niches

The innate immune system of fish is considered first line of defense against a broad spectrum of pathogens. Being a component of innate immunity and lying at the interface between fish and the aqueous environment, skin mucus plays a frontier role in protecting fish from infections. In the present study, skin mucus of Cirrhinus mrigala, Labeo rohita, Catla catla, Rita rita and Channa punctata, inhabiting different ecological niches, was analyzed to characterize potential innate immune factors such as lysozyme, proteases, phosphatases, esterase and sialic acid. The enzyme activities were high in bottom dweller species, C. punctata and C. mrigala, and low in clean water inhabiting species, L. rohita and C. catla. An inverse relationship was observed between the level of enzyme activity and the sialic acid content in these fish species. In R. rita, however, the levels of all factors were found to be low. Zymographic analysis with labeled Micrococcus lysodeikticus revealed three isoforms of lysozyme in C. punctata and two in each species, C. mrigala, L. rohita and C. catla. In R. rita, lysozyme could not be detected. Gelatin zymography revealed that serine and metalloproteases were the major mucus proteases in all fish species investigated. In addition, trypsin-like protease and Ca++-specific serine proteases were observed in skin mucus. Increased knowledge of these parameters could be useful in understanding the role of skin mucus in the innate immune system of fish species inhabiting different ecological niches.

Morphological specializations of the buccal cavity in relation to the food and feeding habit of a carp Cirrhinus mrigala: A scanning electron microscopic investigation

The buccal cavity of an herbivorous fish, Cirrhinus mrigala, was investigated by scanning electron microscopy to determine its surface ultrastructure. The buccal cavity shows significant adaptive modifications in relation to food and feeding ecology of the fish. The buccal cavity of the fish is of modest size and limited capacity, which is considered an adaptation with respect to the small‐sized food items primarily consumed by the fish that could be accommodated in a small space. Modification of surface epithelial cells, on the upper jaw, into characteristic structures—the unculi—is considered an adaptation to browse or scrap, to grasp food materials, e.g., algal felts, and to protect the epithelial surface against abrasions, likely to occur during their characteristic feeding behavior. Differentiation of the highly specialized lamellar organ on the anterior region of the palate could be an adaptation playing a significant role in the selection, retention, and sorting out of palatable food particles from the unpalatable items ingested by the fish. The filamentous epithelial projections and the lingulate epithelial projections on the palatal organ in the posterior region of the palate are considered to serve a critical function in final selection, handling, maneuvering, and propelling the food particles toward the esophagus. The abundance of different categories of taste buds in the buccal cavity suggests that gustation is well developed and the fish is highly responsive in the evaluation and the selection of the preferred palatable food items. The secretions of mucous cells in the buccal cavity are associated with multiple functions—particle entrapment, lubrication of the buccal epithelium and food particles to assist smooth passage of food, and to protect the epithelium from possible abrasion. These morphological characteristics ensure efficient working of the buccal cavity in the assessment of the quality and palatability of ingested food, their retention and transport toward the esophagus. Such an adaptation may be essential in conducting the function most basic to the survival of the individuals and species—feeding. J. Morphol. 2009.

Histochemical analysis of glycoproteins in the secretory cells in the gill epithelium of a catfish, Rita rita (Siluriformes, Bagridae).

Glycoproteins (GPs) were visualised histochemically in the secretory cells - the mucous goblet cells (the type A and the type B), the serous goblet cells, the club cells and the epithelial cells in the gill epithelium of Rita rita. The type A mucous goblet cells, the type B mucous goblet cells and the epithelial cells elaborate GPs with oxidizable vicinal diols and GPs with sialic acid residue without O-acyl substitution. In addition, GPs with O-sulphate esters are elaborated by the type A and GPs with O-acyl sugars by the type B mucous goblet cells. GPs are absent in the serous goblet cells and are with oxidizable vicinal diols in low moieties in the club cells. The analysis of the results elucidates interesting differences in the composition and concentration of GPs in the mucus elaborated by the epithelium of the gill arches and the gill rakers; and the gill filaments and the secondary lamellae indicating the potential importance of the glycoproteins at these locations. GPs elaborated on the surfaces of the gill arches and the gill rakers could be associated to assist in feeding activities and on the surfaces of the gill filaments and the secondary lamellae in the respiratory activity.

Characterisation of glycoproteins in the secretory cells in the operculum of an Indian hill stream fish Garra lamta (Hamilton) (Cyprinidae, Cypriniformes)

Glycoproteins (GPs) elaborated by the secretory cells in the opercular epidermis (OE) and the epithelium lining the inner surface of the operculum (EISO), of an Indian hill stream fish Garra lamta have been analysed by means of a battery of histochemical methods. These included methods for the characterisation and simultaneous visualisation of GPs with oxidizable vicinal diols, O-acyl sugars, O-sulphate esters and sialic acid residues without O-acyl substitution or with O-acyl substitution at C7, C8 or C9. Results obtained in this study show that conventional periodic acid Schiffs and alcian blue based procedures are less sensitive and largely fail to identify different classes of GPs. In the OE the secretory cells involved in the synthesis of GPs are the epithelial cells, the mucous cells and the club cells. Based on the histochemical characterisation of GPs, the mucous cells are distinguished in to two types, Type A and Type B. The majority being of Type A. In the EISO, in contrast, the club cells are absent and most mucous cells belong to a third category, Type C. Type A and Type B mucous cells are few. GP classes synthesised by the secretory cells and released on the outer surface of the operculum, directly exposed to the environmental hazards show significant differences in their composition and concentration from those on, the relatively well protected, inner surface of the operculum. These are discussed in relation to the physiological significance of the GP classes with special reference to their role in lubrication, protection and inhibition of the invasion and proliferation of pathogenic micro-organisms.

Glycoproteins in the epithelium of lips and associated structures of a hill stream fish Garra lamta (Cyprinidae, Cypriniformes): a histochemical investigation.

A series of histochemical procedures were employed to localize and characterize glycoprotein (GP) classes elaborated in the epithelia of the upper and lower lips and associated structures, namely the rostral cap, the adhesive pad, the horny upper and lower jaw sheaths and the folds of skin between them, of a hill stream fish Garra lamta. The epithelia of the lips, the folds of skin and the major portions of the rostral cap and the adhesive pad are mucogenic. The epithelia of the horny jaw sheaths and parts of the rostral cap and the adhesive pad are keratinized. Based on the histochemical characterization of GPs, the cells involved in the secretions in the epithelia at the mucogenic regions of the rostral cap and the adhesive pad comprise the epithelial cells, the type A mucous cells and the club cells. In the lips and the folds of skin, in contrast, the club cells are absent and most mucous cells belong to the type B category. Type A mucous cells are few. GPs elaborated by cellular components of the mucogenic epithelia include GPs with oxidizable vicinal diols, GPs with O‐sulphate esters, GPs with sialic acid residues without O‐acyl substitution or with O‐acyl substitution at C7, C8 or C9 and GPs with O‐acyl sugars. The different types of cells show significant differences in the classes as well as in the concentrations of the GPs elaborated by them. GPs have also been identified in the subcorneal space between the unculi and the epithelial cells in the replacement layer in the epithelia at the keratinized regions. Elaboration of more than one type of GPs suggests a basis for functional discrimination in their role in the mucous secretions at the surface as an adaptation to the feeding ecology and the environment inhabited by the fish.

Functional organization of the skin of the ‘Green-puffer fish’Tetraodon fluviatilis (Ham.-Buch.) (Tetraodontidae, Pisces)

SummaryThe present study concerns the functional organization of the skin ofTetraodon fluviatilis. The epidermis consists of five different types of cells — the flask-shaped mucous cells, the eosinophilic granular cells, the sacciform granulated cells, the vesicle containing granulated cells, and the polygonal cells. A thin noncellular layer, the cuticle found on the surface of the skin, is probably secreted from the polygonal cells in the outermost layer of the epidermis. A,well-defined lymphatic plexus exists between the cells of the basal layer.Numerous triradiate calcareous spines are embedded within elastic connective tissue pockets in the thick dermis. These pockets are filled with an amorphous, acellular, PAS positive material, and are richly supplied with fine blood capillaries. A histomorphologic basis for the erection of the spines and various structural modifications in the skin facilitating its enormous stretching under inflated conditions of the fish are discussed.

Surface ultrastructure of gill arches and gill rakers in relation to feeding of an Indian major carp, Cirrhinus mrigala

The surface ultrastructure of the gill arches and the gill rakers of an herbivorous fish, Cirrhinus mrigala was investigated by scanning electron microscopy. These structures show significant adaptive modifications associated with the food and feeding ecology of the fish. Closely lying short gill rakers and narrow inter-raker channels on the gill arches are associated to filter and retain food particles. Prominent epithelial protuberances on the gill rakers and the gill arches enable the taste buds, located at their summit, to project well above the surface of the epithelium. This could increase the efficiency of the taste buds in selective sorting of palatable food. Surface specializations of the postlingual organ are recognized adaptive modifications for selecting, trapping or holding food particles. Prominent molariform teeth born on the lower pharyngeal jaw, and the chewing pad opposite it, are associated to work together as an efficient pharyngeal mill. Mucous goblet cells are considered to elaborate mucus secretions to trap, glue and lubricate food particles for their smooth transport for swallowing.

Structure and Histochemistry of the Epithelia of Lips and Associated Structures of a Catfish Rita rita

InRita rita the upper lip is associated with the rostral cap and the lower lip with a skin fold. The epithelia of the lips, which are modified with respect to the peculiar feeding behaviour of the fish, are mucogenic. The superficial layer of epithelial cells, in addition to the mucous cells, are involved in active secretion of mucopolysaccharides. Mucus, apart from its diverse functions, as in fish epidermis, lubricates the epithelia giving protection against possible mechanical injury during the searching and catching of prey from bottom debris. Furthermore, the epithelia are thick, an adaptation to their mucogenic nature, so as to provide additional protection. The contents of the club cells are proteinaceous, which correlates with the helical filaments reported in similar cells in fish epidermis. Characteristic ridges on the surface of the lips are considered to be an adaptation to assist in manipulation during the procuring of food. Mucus secreted in the grooves between them reduces friction and prevents the deposition of foreign matter. The skin fold facilitates an increase in gape. The taste buds and ampullary organs, which on the lips and the rostral cap are associated with the localization of prey and triggering of a pick-up reflex, are not located on the skin fold, as the latter often remains concealed between the lower lip and the ventral head skin. The importance of histochemical analysis as a supplement to histological studies is emphasised.

The epidermal and inner epithelial lining of the operculum in Clarias batrachus (Clariidae, siluriformes)

The operculum may be divided into the proximal, the distal and the intermediate regions. The epithelium lining the inner surface of the operculum (EISO) and the opercular epidermis (OE) of these regions inClarias batrachus show significant differences in their thickness, in the density, in the dimensions of mucous cells and club cells, and in the distribution of lymphocytes, melanocytes, taste buds and ampullary organs. These differences in structural organization are associated with the different conditions prevailing at these locations. Rich vascularization observed in the tissues underlying the OE has been correlated with assisting the fish in accessory respiration. In contrast, the tissues underlying the EISO are poorly vascularized. Accessory respiration in this region may not be so advantageous.

The First Evidence of Cholinesterases in Skin Mucus of Carps and its Applicability as Biomarker of Organophosphate Exposure

The presence of cholinesterase (ChE) activity in skin mucus of three carps, Cirrhinus mrigala, Labeo rohita, and Catla catla and its applicability as biomarker of the organophosphorus insecticide exposure were investigated. Biochemical characterization, using specific substrates and inhibitors, indicated that measured esterase activity in skin mucus was mainly owing to ChEs. Significant difference in the proportion of acetylcholinesterase and butyrylcholinesterase activities was observed in skin mucus of three carps. Enzyme kinetic analysis, using the substrate acetylthiocholine iodide revealed significantly high Vmax value in C. catla compared to that in L. rohita and C. mrigala. In contrast, Vmax value using the substrate butyrylthiocholine iodide was significantly high in C. mrigala than in L. rohita and C. catla. In vitro treatment of skin mucus of three carps, with the organophosphorus insecticide Nuvan®, showed strong inhibition of ChE activities. In vivo experiments conducted using C. mrigala and exposing the fish to the sublethal test concentrations (5 and 15 mg/L) of the insecticide also revealed significant inhibition of ChE activity in mucus. In C. mrigala, exposed to the sublethal test concentrations of the insecticide for 4 days and then kept for recovery for 16 days, mucus ChE activity recovered to the control level. Thus, ChE activity in skin mucus could be considered a good biomarker of the organophosphorus insecticide exposure to fish and a useful tool in monitoring environmental toxicity.