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Postnatal ecdysis establishes the permeability barrier in snake skin: new insights into barrier lipid structures

M. C. Tu^1,*, H. B. Lillywhite^1,, J. G. Menon² and G. K. Menon³

¹ Department of Zoology, University of Florida, Gainesville, FL 32611-8525, USA
² Department of Biology, William Paterson University of New Jersey, Wayne, NJ 07470, USA
³ Department of Ornithology and Mammalogy, California Academy of Sciences, Golden Gate Park, San Francisco, CA 94118, USA
^* Present address: Department of Biology, National Taiwan Normal University, Taipei, Taiwan 116, Republic of China

View larger version (16K): [in a new window]   Fig. 1. Diagram of apparatus used in measurements of evaporative water loss from hatchling king snakes. RH, relative humidity.

View larger version (10K): [in a new window]   Fig. 2. Changes in mean total body length (±2 S.E.M.) measured in 20 hatchling California king snakes (Lampropeltis getula) during the course of two postnatal shedding cycles. Each measurement was made at the time of TEWL measurements (see Fig. 3), indicated as trial number on the abscissa. Arrows indicate relative timing of the first (E1) and second (E2) ecdysis with respect to trial number. Repeated-measures ANOVA followed by Bonferroni post hoc tests indicate that body length increased significantly from birth to second ecdysis (P<0.0001), while changes in length between the first and second ecdysis (trials 2 and 3) were not significant (P=0.6715).

View larger version (14K): [in a new window]   Fig. 3. Rates of transepidermal water loss (TEWL) and skin resistance (Rs) measured in the same 20 hatchling king snakes (Lampropeltis getula) as in Fig. 2, during four consecutive trials. Values are means ± 2 S.E.M., and arrows indicate the relative timing of first (E1) and second (E2) ecdysis with respect to trial number. ANOVA followed by post hoc tests indicate that mean TEWL measured in trial 1 is significantly greater than the values obtained in subsequent trials (P<0.0001). Similarly, there was a significant increase in Rs following the first trial (P<0.0001) and a second increase following trial 3 (P=0.0022), whereas measurements of Rs during trials 2 and 3 were not statistically different (P=0.1655). The pattern of changes suggests that the mechanism producing changes in Rs is related to ecdysis.

View larger version (17K): [in a new window]   Fig. 4. Mean values of skin resistance (± 2 S.E.M.) measured in each clutch of hatchling king snakes before (b) and after (a) the first postnatal ecdysis, identified by numbers and letters on the abscissa. ANOVA followed by post hoc tests indicate there are no differences among clutch means, either before or after ecdysis (all P>0.07).

View larger version (21K): [in a new window]   Fig. 5. Skin resistance in hatchling king snakes measured before (circles) and after (squares) the first postnatal ecdysis. The post-shed measurement is shown directly above the pre-shed measurement for each individual snake. Note that the pattern of variation among individuals is generally similar before and after ecdysis.

View larger version (11K): [in a new window]   Fig. 6. Temperature differences between skin surfaces of snakes and the chamber (flow-through) air at the time of TEWL measurements (see Fig. 2) in 20 hatchling king snakes. Each value is the mean ± 2 S.E.M.; arrows denote the relative timing of the first (E1) and second (E2) ecdysis with respect to trial number. The measured temperature differential decreases significantly following the second measurement trial (ANOVA followed by post hoc tests, P=0.0076) and correlates with the assimilation of residual yolk (see text).

View larger version (182K): [in a new window]   Fig. 7. (A) Ultrastructure of hatchling skin, sampled on the day of hatching and before the first ecdysis, showing mesos (m), (a) and ß (b) layers above the germinative layer (g, granular layer). Inset shows semi-thick plastic section (0.5-1 µm) of the same. (B) Post-shed skin sampled 2 days after the first ecdysis shows increased thickness in all three layers at the same magnification as in A. Note the near doubling of cells in the mesos layer. The germinative layer appears more compact compared to A. Inset shows light microscopic features of the post-shed skin in semi-thick plastic section (0.5-1 µm). The gaps in mesos layers seen in the micrographs are artifacts in tissue preparation (OsO4 post-fixation). Scale bars, 1.0 µm; in insets, 0.1 µm.

View larger version (166K): [in a new window]   Fig. 8. (A) Mesos layer (m) showing somewhat disorganized bilayer structures in the intercellular spaces (arrows) in pre-shed skin sampled on the day of hatching. (B) High magnification ultrastructure of the layer in pre-shed skin of same hatchling showing a desmosome (d) and lamellar lipid inclusions (ll) in the outer (a) layer. Such an abundance of lipid is not seen in the section of inner cells (ia), shown here, which exhibits an abundance of membrane structures resembling trans-Golgi (RuO4 post-fixation). Scale bars, 0.1 µm. Inset: a frozen section (10-12 µm) of skin stained with Fat Red-7B showing presence of neutral lipids, but not well demarcated compared to that in Fig. 9 inset of post-shed skin. Scale bar for inset, 10 µm.

View larger version (152K): [in a new window]   Fig. 9. (A) Mesos layer (m) in post-shed skin (2 days) showing well-organized and continuous bilayers in intercellular domains (arrows). (B) High magnification ultrastructure of the layer in the same post-shed skin (RuO4 post-fixation). Note decreased lamellar lipid inclusions (ll) as well as electron-lucent lipid inclusions (1) in the outer cell compared to the pre-shed skin in Fig. 8. Desmosomes (d) are clearly seen in this field. Scale bars, 0.1 µm. The inset shows light microscopic histochemistry of frozen section (10-12 µm) stained for neutral lipids. Note the improved staining in the stratum corneum compared with Fig. 8, inset. Scale bar for inset, 10 µm.

View larger version (181K): [in a new window]   Fig. 10. Higher magnification electron micrograph of a portion of the inner (a) cell in pre-shed skin showing lipid inclusions including a multigranular lamellar body (mlb), large lamellar (ll) and electron-lucent lipid (l) inclusions (RuO4 stain) in close association with elements of the tubulo-reticular membrane system (arrow). This skin was sampled on the day of hatching, as in Figs 7 and 8. Inset: avian multilamellar bodies at comparable magnification, to highlight the structural similarity to the snake organelle. Scale bars, 0.1 µm.