COUNTS OF GROWTH LAYER GROUPS IN CEMENTUM AND DENTINE IN RINGED SEALS (PHOCA HISPIDA)

We compared counts of growth layer groups (GLGs) in the dentine of un-decalcified, unstained cross-sections and in the cementum of decalcified, stained longitudinal sections of canine teeth from 144 ringed seals ( Phoca hispida ). Although there was a statistically significant correlation until approximately 10 GLGs, about 75% of paired readings at ≤ 10 cementum GLGs disagreed. After 10 GLGs, the number of GLGs in the cementum usually was greater. The maximum GLG count in cementum was 33, compared to a maximum in dentine of only 19. Interobserver differences in median counts were not statistically significant using cementum or dentine counts. Regression analysis revealed that for cementum in female seals, readers differed at higher counts (P < 0.05), and for dentine, there was a constant difference of about 0.6 GLGs ( P < 0.05) for male seals and 1.1 GLGs ( P < 0.05) for female seals. Counting GLGs in the cementum of decalcified and stained longitudinal sections provided higher counts and more agreement between readers, and it was the better of the methods examined for ageing ringed seals.     

On the nature of the opaque and translucent enamel regions of some macropodinae (Macropus giganteus,Wallabia bicolor and Peradorcas concinna)

Summary Teeth of three macropod species, M. giganteus, W. bicolor and P. concinna, have been studied using the techniques of light microscopy, scanning- and transmission-electron microscopy and hardness measurement. Light microscope observations showed that the teeth of these species had a translucent enamel region close to the dentine and an outer opaque enamel region at the tooth's surface. These regions were not related to the presence or absence of tubules which are a characteristic feature of marsupial enamel. Hardness tests showed that the opaque enamel was softer than the translucent enamel. Scanning electron microscope observations revealed that there was no correlation between any particular prism packing or orientation and the opaque and translucent enamel regions. Transmission electron microscope observations showed that the translucent enamel region consisted of well defined prisms and well packed, lath-like crystals, whereas the opaque enamel was disrupted by voids (which ranged in size from enlarged micropores to about 2 m in diameter in extreme cases) between crystals and some randomly oriented, loosely packed crystals. This disruption within the opaque enamel region was more common at prism boundaries but pockets of disrupted enamel were also found within prisms and interprismatic regions. The opacity of the enamel was caused by scattering of light from the voids. The ultrastructure of the opaque enamel region indicated that this region was hypomineralized; hardness tests and polarized light microscope observations were consistent with these results.     

EVALUATION OF AN EFFECTIVE METHOD TO ESTIMATE AGE OF CAPE FUR SEALS USING GROUND TOOTH SECTIONS

Teeth of known-age Cape fur seals Arctocephalus pusillus pusillus were used to validate age estimated from ground sections. In the canines, dentine growth layer groups (GLGs) reflected age accurately but no reliable readings could be obtained from GLGs in the cementum. Upper canines were the most suitable for age estimation. By contrast, in the postcanines where the cementum is thicker, only GLGs in the cementum could be used for age determination, but not with the same accuracy as for dentine in the canines. Therefore, it is recommended that GLGs in the dentine be used to determine age in the Cape fur seal. However, pulp cavities in canines closed at about 13 yr and consequently GLGs in the cementum of the postcanines should be used where the pulp cavities of canines are closed. Accurate estimation of age is not possible from the dentine of older animals.     

Comparison of two histological techniques for age determination in small cetaceans

Age estimation in odontocetes is based on counts of growth layer groups (GLGs) deposited in recording structures such as teeth. Generally, tooth sections are obtained using a cryostat microtome. However, some researchers prefer obtaining thin sections using a traditional paraffin microtome. Little information is available on the application of this technique to dolphin teeth. Our main aim was to investigate if the paraffin technique can be a viable alternative. We considered whether estimated age would be affected by preparation technique, staining method, and section thickness, while controlling for effects of species, body length, and sex. We also analyzed whether the staining method would affect readability of GLGs and age reading variability. Teeth from 86 individuals (representing seven species) were used, but not all were prepared using both techniques because sufficient teeth were not available in all cases. Although the staining method had significant effects on the estimated age using both techniques, the variability of GLG counts was small and appeared to be similar for both techniques. Using Mayer's hematoxylin stained sections of 8 μm thickness, good agreement of ages was obtained from both techniques, with more preparations classified as "good quality" for the paraffin technique. Mayer's hematoxylin provided the best contrast of the GLGs when using the paraffin technique. We conclude that the paraffin technique is viable and represents a cost-effective alternative to a cryostat microtome when preparing cetacean teeth for age determination.     

Growth Zones in the Echinoid Skeleton

Growth zones in echinoid skeletal ossicles are mainly the resultof differences in structural characteristics. In the test plates,opaque zones, which appear light in reflected light, dark intransmitted light, and are X-ray dense, have relatively largertrabecules and smaller intertrabecular channels. Translucentzones, which appear dark in reflected light, light in transmittedlight, and are less X-ray dense, have relatively smaller trabeculesand larger intertrabecular channels. Organic material in theplates, especially when pigmented or charred, enhances the appearanceof the growth zones. Opaque zones result from relatively fastplate growth while translucent zones result from relativelyslow plate growth; food deprivation leads to the formation oftranslucent zones. The growth zones appear to be formed seasonally,at least in some cases, probably in relation to seasonal changesin growth rates, and perhaps in relation to seasonal reproductiveactivity     

L’ambre associé aux lignites cénomaniens du Sarladais (Dordogne,SO France)

The mines exploiting the Cenomanian lignites at Simeyrols (Dordogne, France) were long known in geological terms. The presence of amber had been occasionally reported. Recent investigations have helped to collect material from cuttings of the old exploitations that provided amber beads of small size. The grains have mostly a teardrop shape more or less elongated and appear translucent. The opaque parts of the grains correspond to colonization of filamentous microorganisms that will be further studied.     

Age estimation in bowhead whales using tympanic bulla histology and baleen isotopes

Tympanic bullae and baleen plates from bowhead whales of the Western Arctic population were examined. Growth layer groups (GLGs) in the involucrum of the tympanic bone were used to estimate age of the whales, and compared to stable isotope signatures along transects of baleen plates and the involucrum. The involucrum of the tympanic bone consists of three regions that form in utero, during nursing in the first year, and during the first decades of life, respectively. Life history events, such as annual migration, are recorded in the bowhead tympanic bulla. It is likely that bone growth in the bowhead tympanic occurs during periods of high food intake, while slow or arrested growth occurs during periods of low food intake. Comparisons between numbers of GLGs in the tympanic, number of isotopic oscillations in a baleen plate, length of the baleen plate, and total whale length show correlation coefficients as high as 0.97. The tympanic GLG method is particularly useful for estimating the age of whales up to 20 yr old.     

STRUCTURAL,CHEMICAL AND ECOLOGICAL STUDIES ON IRIDESCENCE IN IRIDAEA (RHODOPHYTA)1

Transmission electron microscopy of the iridescent algae Iridaea flaccida (S & G) Silva, Iridaea cordata (Turn.) Bory var. cordata and I. cordata var. splendens (S & G) Abbott reveals a multilaminated cuticle covering the thallus. Experimental results show the cuticle: a) can be isolated intact by mechanical scraping or NaOH treatment; b) is iridescent by itself and the denuded thallus is not; and, c) is isolated without any subtending polysaccharide layer, cell walls, or cells. This cuticle acts as a thin layer producing the constructive and destructive light interference which is seen as iridescence. It is formed of alternating electron opaque and translucent layers with a total thickness of 0.5–1.6 μm. Analysis of mechanically isolated cuticle shows that it is composed of protein (50%), carbohydrate (ca. 40%), inorganic salts (5%) and some fatty acids (less than 1.0%). The electron opaque layers may correspond to protein-rich regions and the electron translucent ones to regions rich in carbohydrates. The cuticle does not appear to affect photosynthesis or respiration, but rather, may protect the alga from physical factors such as desiccation and from predator injury. It is likely that the iridescence in other foliaceous red algae is caused by a similar structure.     

Factors influencing in vitro killing of bacteria by hemocytes of the eastern oyster (Crassostrea virginica).

A tetrazolium dye reduction assay was used to study factors governing the killing of bacteria by oyster hemocytes. In vitro tests were performed on bacterial strains by using hemocytes from oysters collected from the same location in winter and summer. Vibrio parahaemolyticus strains, altered in motility or colonial morphology (opaque and translucent), and Listeria monocytogenes mutants lacking catalase, superoxide dismutase, hemolysin, and phospholipase activities were examined in winter and summer. Vibrio vulnificus strains, opaque and translucent (with and without capsules), were examined only in summer. Among V. parahaemolyticus and L. monocytogenes, significantly (P < 0.05) higher levels of killing by hemocytes were observed in summer than in winter. L. monocytogenes was more resistant than V. parahaemolyticus or V. vulnificus to the bactericidal activity of hemocytes. In winter, both translucent strains of V. parahaemolyticus showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. In summer, only one of the V. parahaemolyticus translucent strains showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. No significant differences (P > 0.05) in killing by hemocytes were observed between opaque (encapsulated) and translucent (nonencapsulated) pairs of V. vulnificus. Activities of 19 hydrolytic enzymes were measured in oyster hemolymph collected in winter and summer. Only one enzyme, esterase (C4), showed a seasonal difference in activity (higher in winter than in summer). These results suggest that differences existed between bacterial genera in their ability to evade killing by oyster hemocytes, that a trait(s) associated with the opaque phenotype may have enabled V. parahaemolyticus to evade killing by the oyster's cellular defense, and that bactericidal activity of hemocytes was greater in summer than in winter.     

Factors Influencing In Vitro Killing of Bacteria by Hemocytes of the Eastern Oyster (Crassostrea virginica)

A tetrazolium dye reduction assay was used to study factors governing the killing of bacteria by oyster hemocytes. In vitro tests were performed on bacterial strains by using hemocytes from oysters collected from the same location in winter and summer. Vibrio parahaemolyticus strains, altered in motility or colonial morphology (opaque and translucent), and Listeria monocytogenes mutants lacking catalase, superoxide dismutase, hemolysin, and phospholipase activities were examined in winter and summer. Vibrio vulnificus strains, opaque and translucent (with and without capsules), were examined only in summer. Among V. parahaemolyticus and L. monocytogenes, significantly (P < 0.05) higher levels of killing by hemocytes were observed in summer than in winter. L. monocytogenes was more resistant than V. parahaemolyticus or V. vulnificus to the bactericidal activity of hemocytes. In winter, both translucent strains of V. parahaemolyticus showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. In summer, only one of the V. parahaemolyticus translucent strains showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. No significant differences (P > 0.05) in killing by hemocytes were observed between opaque (encapsulated) and translucent (nonencapsulated) pairs of V. vulnificus. Activities of 19 hydrolytic enzymes were measured in oyster hemolymph collected in winter and summer. Only one enzyme, esterase (C4), showed a seasonal difference in activity (higher in winter than in summer). These results suggest that differences existed between bacterial genera in their ability to evade killing by oyster hemocytes, that a trait(s) associated with the opaque phenotype may have enabled V. parahaemolyticus to evade killing by the oyster’s cellular defense, and that bactericidal activity of hemocytes was greater in summer than in winter.     

The utility of otolith microstructure in determining the timing and position of the first annulus in juvenile Antarctic toothfish (<Emphasis Type="Italic">Dissostichus mawsoni</Emphasis>) from the South Shetland Islands

Juvenile Antarctic toothfish (Dissostichus mawsoni) collected off the South Shetland Islands (CCAMLR Subarea 48.1) were aged by counting the presumed daily increments in the sagittal otoliths. The timing and position of the first annulus was examined for each individual fish from 10 to 13 cm TL. Under reflected light at low magnification, the otolith sections exhibited a wide opaque nucleus, with no evidence of deposition of the first translucent zone. The average measurement of ventral and dorsal radii of these sections were, respectively, 603 and 768 μm, consistently less than the measurements done in previously estimated 1 year old fish caught in the same area. All specimens aged in the present study were approximately 1 year old fish, with an age range between 365 and 431 days, suggesting a significant underestimation of age in the previous studies. The value of ageing precision indices calculated was relatively low, supporting the reliability of the reading technique applied. Two checks were easily identified in the core of all otolith sections, interpreted tentatively as hatching and first exogenous feeding checks. Back-calculating from the time of capture, the hatching period of D. mawsoni was estimated to occur throughout the summer season, from November to February. In conclusion, the delay in timing of deposition of the first translucent zone, which occurs in the second winter from hatching, should be taken into account in future studies on ageing Antarctic toothfish, as well as in the stock assessment models based on length at age data.     

Plasmid deoxyribonucleic acid and translucent-to-opaque variation in Mycobacterium intracellulare 103.

After treatment with mitomycin D and other antibacterial agents, a translucent, smooth-colony-forming mycobacterium, isolated from sputum and designated as Mycobacterium intracellulare strain 103, gave rise to variants forming opaque colonies. These opaque variants were more sensitive streptomycin, kanamycin, viomycin, and rifampin than were the wild-type translucent variants. Plasmid deoxyribonucleic acids taken from translucent strain cells and from cells of certain opaque variants were analyzed by agarose gel electrophoresis. Two plasmids of molecular weights of approximately 2 x 10(6) and 50 x 10(6), respectively, were found in the wild-type translucent cells; one of them, the 2 x 10(6)-molecular-weight plasmid, was always missing from deoxyribonucleic acids of the opaque variant cells. The results suggested that translucent colonial appearance and antibiotic resistance of the strain are plasmid-determined functions.     

Viability of Vibrio vulnificus in Association with Hemocytes of the American Oyster (Crassostrea virginica)

Certain indigenous estuarine bacteria, such as Vibrio vulnificus, may cause opportunistic human infections after consumption of raw oysters or exposure of tissues to seawater. V. vulnificus is known to be closely associated with oyster (Crassostrea virginica) tissues and is not removed by controlled purification methods, such as UV light-assisted depuration. In fact, when live shellfish are subjected to controlled purification, the number of V. vulnificus cells can markedly increase. A review of previous studies showed that few workers have examined mechanisms in oysters which may influence the persistence of V. vulnificus in shellfish, such as the fate of V. vulnificus following phagocytosis by molluscan hemocytes. The objectives of this study were to define the intracellular viability and extracellular viability of V. vulnificus during the phagocytic process and to study the release of specific lysosomal enzymes. The viability of a virulent estuarine V. vulnificus isolate with opaque morphology was compared with the viability of a translucent, nonvirulent form, the viability of Vibrio cholerae, and the viability of Escherichia coli in phagocytosis experiments. Our results showed that the levels of phagocytosis and bactericidal degradation of the opaque V. vulnificus isolate were less than the levels of phagocytosis and bactericial degradation of the translucent morphotype. These findings indicate that encapsulation may contribute to resistance to ingestion and degradation by hemocytes. The rates of intracellular death of V. cholerae and E. coli exceeded the rate of intracellular death of the opaque V. vulnificus isolate, even though the ingestion or uptake rates did not differ significantly. The levels of lysozyme activity and acid phosphatase activity were not significantly different in hemocyte monolayers inoculated with V. vulnificus.     

Aspects of the Life History of the Salema, Sarpa salpa (Pisces, Sparidae), off the Canarian Archipelago (Central-East Atlantic)

Life history of the salema, Sarpa salpa, off the Canarian archipelago was studied from samples collected between January 1998 and December 1999. Fish ranged from 119 to 452 mm in total length and from 24.9 to 1491.7 g in weight. The species was characterized by a protandric hermaphroditism. The overall sex ratio was unbalanced in favour of males (1 : 0.41). The reproductive season extends from September to March, with a peak in spawning activity in December–January. Males reached maturity at a smaller length (226 mm, 2 years old) than females (294 mm, 3 years old). Males, females and all fish showed an isometric growth. The morphometric relationship between length and mass for the whole population was described by the parameters: a = 0.0000134 and b = 3.01. A concentric pattern of opaque and translucent zones was readily distinguishable on the otoliths. Two rings, one opaque and one translucent, were laid down each year on the otoliths; the opaque zone was formed during the summer months, and the translucent zone during the winter months. Individuals aged 0 to 11 years were found. The parameters of the von Bertalanffy growth equation for all individuals were: L = 479 mm, k = 0.212 year^–1, and t₀ = –1.08 year.     

ELECTRON MICROSCOPY OF THE AVIAN RENAL GLOMERULUS

Electron microscopy of sections of chicken glomeruli shows them to possess a large central cell mass, occupying the hilum and the centre of the glomerulus, and continuous with the adventitia of the afferent and efferent arterioles. The glomerular capillaries form a much simpler system than in mammals and are spread over the surface of the central cell mass. Between the capillaries the mass is limited externally by the major component of the glomerular capillary basement membrane, which continues over the surface of the mass from one capillary to the next. Projections of the central cell mass characteristically form the support for glomerular capillaries, and smaller knobs of the central mass may project actually into the lumen of the capillaries, but always carry a layer of endothelial cytoplasm before them. They are never in direct contact with blood. The basement membrane of the glomerular capillary loop has a central dense layer and two lateral less dense layers as in mammals. The central dense layer is continuous with similar appearing dense material in the intercellular spaces of the adventitiae of the arterioles, and also with that of the central cell mass. The two less dense layers can also be traced into direct continuity with the less dense regions of this intercellular substance. The endothelial cytoplasm is spread as a thin sheet over the inner surface of the capillary basement membrane, and shows scattered "pores" resembling those described in mammals. Epithelial cells with interlacing pedicels are at least as prominent as those in mammals. Bowman's capsular membrane also possesses three layers similar to but less wide than those of the capillary basement membrane, and all three layers can be traced into continuity with the dark and light regions of the intercellular material of the adventitial cells of the arterioles, and beyond them with that of the central cell mass. At the hilum Bowman's capsular membrane also fuses with the capillary basement membrane.     

Annual variability in dentin δ15N and δ13C reveal sex differences in weaning age and feeding habits in Risso's dolphins (Grampus griseus)

Teeth of odontocetes accumulate annual dentinal growth layer groups (GLGs) that record isotope ratios, which reflect the time of their synthesis. Collectively, they provide lifetime records of individual feeding patterns from which life history traits can be inferred. We subsampled the prenatal dentin and postnatal GLGs in Risso's dolphins (Grampus griseus) (n = 65) that stranded or were collected as bycatch in Taiwan (1994–2014) and analyzed them for δ¹⁵N and δ¹³C. Age‐specific δ¹⁵N and δ¹³C values were corrected for effects of calendar year, stranding site, C/N, and sex. δ¹⁵N values were higher in prenatal layers (14.94‰ ± 0.74‰) than in adult female GLGs (12.58‰ ± 0.20‰), suggesting fetal enrichment during gestation. Decreasing δ¹⁵N values in early GLGs suggested changes in dietary protein sources during transition to complete weaning. Weaning age was earlier in males (1.09 yr) than in females (1.81 yr). Significant differences in δ¹⁵N values between weaned males and females suggest potential sexual segregation in feeding habits. δ¹³C values increased from the prenatal to the 4th GLG by ~1.0‰, indicative of a diet shift from ¹³C‐depleted milk to prey items. Our results provide novel insights into the sex‐specific ontogenetic changes in feeding patterns and some life history traits of Risso's dolphins.     

Phytochrome: First-order phototransformation kinetics in vivo

Summary The deviation from first order commonly observed in phototransformation kinetics of phytochrome in vivo is due to a light-intensity gradient within the sample. This gradient was measured and was found to approach that predicted by the Kubelka-Munk theory of light scatter in turbid materials. The influence of this gradient is eliminated and first-order phototransformation kinetics are obtained, when either (i) thin samples of translucent (low optical density) material of high phytochrome content are measured directly; or (ii) thin samples of opaque (high optical density) or translucent material are sandwiched between two layers of light-scattering material. This result is consistent with the existence of only one population of photoreversible phytochrome molecules in vivo.     

Fine structural and immunohistochemical localization of cardiac hormones (ANP) in the right atrium and hypothalamus of the white rat

Atrial natriuretic peptide (ANP) is a polypeptide hormone secreted primarily by atrial myoendocrine cells. It has diuretic, natriuretic and vasorelaxant effects. ANP has been characterized by non-morphological methods in a number of extra-atrial tissues, particularly the hypothalamus, but little is known of the immunohistochemistry of hypothalamic ANP cells in comparison to atrial ones. Although the presence of ANP-producing cells has previously been confirmed in the right atrium of the rat and other vertebrate species, to our knowledge, this is the first study to demonstrate the presence of these cells in the hypothalamus using a purely morphological method such as electron microscopy. The fine structural and immunohistochemical characteristics of right atrial and hypothalamic ANP positive cells were investigated using immunogold labeling with goat anti-alpha-human ANP (1-28) as primary antibody. Atrial ANP cells were characterized by the presence of membrane-bound electrondense spherical or oval granules with a diameter of about 250 nm. The opaque content of the granules is separated from the limiting membrane by a thin electron translucent band about 20 nm wide. Electron dense crystalloid inclusions were evident within the granule matrix of some atrial ANP granules. Hypothalamic ANP granules were membrane-bound larger in diameter (320 nm), and less electron dense, and lacked crystalloid inclusions. Statistical analyses revealed a significant larger diameter and a significant smaller number of hypothalamic ANP granules compared to atrial ones. The significantly greater number of atrial ANP positive granules suggests a greater volume capacity for the atrial ANP positive granules as compared to the hypothalamic ones. This may indicate that ANP is secreted primarily from the right atrium and to a lesser extent from the hypothalamus; and that both atrial and hypothalamic ANP are closely related in chemical nature and immunohistochemical characteristics. This supports the suggestion that ANP may play the dual role of peripheral hormone and a neurotransmitter or neuromediator.