Epidermal mucous cells in the chequered water snake Natrix piscator (Colubridae: Squamata). A cytochemical investigation

Mucous cells in the epidermis of Natrix piscator have been discovered using histochemical techniques. They are correlated with the amphibious mode of life of the snake.     

Lipid histochemistry of the epidermis of Natrix piscator during its sloughing cycle

The lipid histochemistry of the scale and hinge epidermis of the chequered water snake, Natrix piscator , throughout the sloughing cycle, has been described. The presence of comparatively high concentrations of phospholipids in the mesos layer and a-layer, in comparison to neutral lipids, has been associated with a permeability barrier to transcutaneous water flux. Free fatty acids, present in almost all epidermal layers and in eosinophilic granular cells, may protect the epidermis from bacterial and fungal attacks. Cholesterol, in addition to phospholipids, in various keratinized layers, is assumed to be derived from membranous structures of epidermal cells and is regarded as a stabilizer of the phospholipids in membranes.     

Circadian rhythmicity in leukocytes immune responses in the freshwater snake,Natrix piscator

Circadian rhythm is observed in most of the physiological functions including immune response. The use of animal models other than mammals is useful in understanding how the vertebrate circadian system is organized and how this biological clock has changed throughout the vertebrate evolution. The present study was aimed to examine the circadian variability in the innate immune responses of leukocytes in the freshwater snake, Natrix piscator. Leukocytes were isolated and processed for total and differential leukocyte count, leukocyte phagocytosis, NBT reduction, nitrite production, and lymphocyte proliferation. Experiments were conducted for seven time points at 24, 4, 8, 12, 16, 20, and 24 h in three seasons – summer, winter, and spring. Cosinor analysis revealed that among leukocytes, only lymphocyte count showed circadian variation in summer. Percent phagocytosis and phagocytic index had significant rhythm of 24 h in winter and summer season, respectively. The acrophase of NBT reduction and nitrite release were coming during the evening hours in summer and during morning hours in winter and had circadian rhythmicity. A significant phase shift in nitrite release was observed with a trend of delayed phase shift from winter to summer. Circadian rhythm was also observed in lymphocyte proliferation (basal and concanavalin A stimulated). It is evident from the present study that animals synchronize their immune activity according to the time of the day and season. Enhancement of immune function helps the individual cope with seasonal stressors that would otherwise jeopardize the survival of animal.     

Carbohydrate histochemistry of the epidermis of Natrix piscator during its sloughing cycle

Carbohydrate histochemistry of the scale and hinge epidermis of the chequered water snake, Nalrix piscator , throughout the sloughing cycle, has been described. The small amount of mucopolysaccharide present in the Oberhautchen, mesos layer, α-layer and β-Mayer (in its initial stage of differentiation) is comparable with that in amphibian epidermis and the epidermis of certain freshwater fish undergoing keratinization. Moderate amounts of mucopolysaccharide in the lacunar tissue and clear layer may protect against environmental pathogens and retain water to protect the epidermis from desiccation. Mucous cells could not be located in the epidermis throughout the sloughing cycle, contrary to some previous observations that they occur in the hinge region. The general absence of glycogen in the epidermis in most stages of the sloughing cycle suggests that the glycogen metabolized in the epidermis is utilized immediately, in view of the high energy requirements of proliferation and differentiation. Accumulation of glycogen granules in the presumptive α-layer in stage 2 and in the clear layer, presumptive Oberhautchen and presumptive β-Mayer in stage 3 is correlated with low energy requirements, indicating a slowing down of the process of keratinization of cells in these layers.     

Serum calcium level of freshwater snake, Natrix piscator, in response to vitamin D3 administration

The effect of i.m. injection of vitamin D3 (25 IU/100 g b.wt) on serum calcium level was investigated in Natrix piscator. This treatment evokes hypercalcemia at day 3 which progresses up to day 5. Thereafter, a decline was observed in the serum calcium level at day 10 and day 15.     

Immune response in the water snake,Natrix erythrogaster transversa

One month after an intramuscular injection of bovine albumin, the plasma of a water snake (Natrix erythrogaster transversa) contained a macroglobulin which bound radiolabeled bovine serum albumin.     

Ultimobranchial body and parathyroid glands of the freshwater snake Natrix piscator in response to vitamin D3 administration.

Vitamin D3 (20 I.U./100 g body wt) was administered to the freshwater snake Natrix piscator for 15 days. Elevation of serum calcium and inorganic phosphate levels was observed after the treatment. The ultimobranchial body became active whereas the parathyroid glands exhibited reduced activity.     

Dietary regulation of digestive enzyme levels in the water snake, Natrix tessellata

Levels of digestive enzymes were analyzed in water snakes following artificial feeding. A prominent increase of total proteolytic activity in the stomach was evident after feeding with a casein solution or after the snake was offered a fish (9- and 6-fold that of the fasting level, respectively). The activity following feeding with starch was much lower. Increased levels of chymotrypsin(ogen) as well as of amylase were evident in the pancreas 1 day after feeding the snake with fish. A specific induction of increased level of chymotrypsin in the pancreas of adult snakes was achieved by feeding with casein (12-fold that of the fasting level). In the group fed with starch, the chymotrypsin level dropped, while a 3-fold increase of amylase was evident. In newborn snakes, fed for the first time, casein induced a dramatic increase in the level of chymotrypsin in the pancreas (58 times the fasting level); feeding with starch induced an approximate 2-fold increase of chymotrypsin. Histological examination of the pancreas 1 day following casein feeding showed acinar cells loaded with zymogen granules. In starved animals and in snakes fed with starch, a much lower concentration of zymogen granules was observed. The pancreas of the snake may, thus, be most suitable for studying the specific induction of synthesis of digestive enzymes.     

Enzymes in the epidermis of Natrix piscator during its sloughing cycle

Acid phosphatase, non-specific esterase, alkaline phosphatase, monoamine oxidase and true lipase activities, in the epidermis of Natrix piscator in different stages of the sloughing cycle, have been localized using various histochemical techniques.
Different layers in scale epidermis have staining properties similar to corresponding layers in hinge epidermis.
Acid phosphatase and non-specific esterase activity in cell layers undergoing keratinization, and the lacunar tissue undergoing disintegration are associated with hydrolytic and catabolic wasting processes involving cell death. The activity of these enzymes in the clear layer is associated with the breaking down of the cementing substance resulting in the separation of clear layer from underlying tissue and facilitating the shedding of old slough.
Alkaline phosphatase activity in the stratum germinativum and undifferentiated epidermal cells has been associated with cell proliferation and differentiation. The presence of alkaline phosphatase in the lacunar tissue and clear layer has been correlated with the synthesis of mucopolysaccharides in these layers.
Monoamine oxidase and true lipase activity could not be located in the epidermis at any stage of the sloughing cycle.     

Daily and Seasonal Rhythms in Immune Responses of Splenocytes in the Freshwater Snake,Natrix piscator

Present study was designed to examine daily and seasonal variability in the innate immune responses of splenocytes in the fresh water snake, Natrix piscator. Animals were mildly anesthetized and spleen was aseptically isolated and processed for macrophage phagocytosis, NBT reduction, nitrite production, splenocyte proliferation and serum lysozyme activity. Samples were collected at seven time points, viz., 0000, 0400, 0800, 1200, 1600, 2000 and 0000 h during three different seasons, namely summer, winter and spring. Cosinor analysis revealed that percent phagocytosis had a significant 24-h rhythm during summer and spring seasons. The peaks of rhythms in NBT reduction and nitrite release occurred in the morning hours at 10.88 h and 8.31 h, respectively, in winter. A significant 24-h rhythm was also observed in lysozyme concentration and splenocyte proliferation (both Basal and Concanavalin A stimulated) in all three seasons. A significant phase shift in splenocyte proliferation was obtained with a trend of delayed phase shift from winter to spring and from spring to summer. Of the nine variables, significant annual (seasonal) rhythms were detected in almost all variables, excluding phagocytic and splenosomatic indices. All rhythmic variables, except spleen cellularity, exhibited tightly synchronized peaks coinciding with the progressive and recrudescence phases of annual reproductive cycle. It is concluded that the snake synchronizes its daily and seasonal immune activity with the corresponding external time cues. The enhancement of immune function coinciding with one of its crucial reproductive phases might be helping it to cope with the seasonal stressors, including abundance of pathogens, which would otherwise jeopardize the successful reproduction and eventual survival of the species.     

Mitochondria with atypical cristae in the hypothalamic neuropil of the water snake Natrix Maura

Two different types of mitochondria with ordered cristae have been identified in the subependymal hypothalamic neuropil of the water snake. Type I mitochondria have bundles of tubular cristae arranged in a hexagonal pattern; the intercristal matrix displays an axial periodicity when sectioned longitudinally and they have only been observed in neuronal processes, mainly in dendrites. Type II mitochondria are typical prismatic-type mitochondria with hexagonally arranged prismatic cristae of triangular profiles, matrix have many dark dots. The functional significance of these mitochondria is still unknown.