Studies on the soft shell membranes of the egg shell of Chelonia mydas L.

The soft membrane fibres of the turtle eggshell like their avian counterparts, consist of an electron dense core surrounded by a less dense mantle. In the oviposited egg the core material is homogeneously electron dense while in oviducal eggs it has a pitted appearance. Histochemical analyses indicate that as in the fowl, the fibres are a protein-carbohydrate complex.     

Studies on the egg shell (oviducal and oviposited) of Chelonia mydas L.

The outer calcified surface of the turtle egg shell consists primarily of crystalline aggregates of calcium carbonate in its aragonite form, together with a small amount (< 5 %) of calcitic material. The latter is first deposited to be followed by aragonite deposition.In the first instance, calcification occurs on the rims of discrete pits formed by the lateral deflection of the ends of soft shell membrane fibres. As crystal deposition continues these pits become filled in and eventually occluded.Micro- and X-ray diffraction analyses of the calcified layer indicate the presence of phosphorus and sulphur. The effects of these elements on the type of crystal deposited, (i.e., aragonite or calcite) is discussed.     

Observations on the histology of the ovary of the Costa Rican green turtle, Chelonia mydas L

A preliminary survey of the ovary of the green turtle, Chelonia mydas L., shows that as in Aves the smaller ovarian follicles have a prominent zona radiata. In this a mucopolysaccharide is deposited eventually forming a thick band located in a position analogous to that of the avian perivitelline membrane but which appears to be impermanent since it is no longer present in the larger follicles (> 10 mm diameter). Its disappearance is accompanied by a marked increase in the height of the granulosa cells which become very tall and accumulate large globules of basic protein and sugar in their apical cytosome while at the same time a similar substance is found in the yolk. The granulosa also seems to disappear later in development leaving the highly developed theca interna in contact with the yolk. The theca is then very vascular and contains large numbers of fat cells. No structure comparable to a corpus luteum has been distinguished but ovarian material obtained within the first few days after ovulation has not been available for study and it is not, therefore, possible to be dogmatic on this question.     

Behavioural studies on the green turtle (Chelonia mydas) in the sea

Underwater observations of green turtles near Fairfax Island, Bunker Group, Australia, disclosed female behaviour patterns to avoid copulation, including a striking adpressed hind limb position; resting in a female sanctuary area; avoidance swimming; face-to-face confrontation, followed or accompanied by biting; and a ‘refusal’ position, with the female assuming a vertical position, her limbs widely outspread and plastron facing the male. Copulation activity precedes egg laying on Fairfax. Copulation lasts as long as 6 hr. Females outnumber males, who engage in multiple copulations. The ‘escort’ is a group of one to five males attendant upon mated pairs. Hatchlings feed on various invertebrates. Cleaner fishes groom adult female turtles.     

Hormonal and metabolic responses to nesting activities in the green turtle, Chelonia mydas

We examined the association between green turtle nesting activities and plasma profiles of hormones that are widely implicated in aspects of heightened metabolism and energy regulation; epinephrine (EPI), norepinephrine (NE) and corticosterone. In conjunction, we examined plasma profiles of glucose and lactate to infer metabolic processes associated with green turtle nesting behaviour. Finally, because these hormones are also involved in mediating behaviour and physiology associated with stressful situations, we examined the effect of a stressor encountered during nesting, physical disturbance, on hormone levels. Plasma profiles of epinephrine, norepinephrine and corticosterone were not significantly altered across different stages of nesting. Plasma glucose and lactate both exhibited significant increases related to nesting activity; glucose increased dramatically during the emergence stage of nesting before stabilizing, and lactate levels continued to increase throughout the nesting process. There was no significant association between plasma hormones and glucose. For female turtles that abandoned nesting activities due to competition for nest space, there was no significant difference in plasma levels of epinephrine, norepinephrine and corticosterone compared to females that persisted with nesting activities. Overall, while distinct metabolic changes took place in nesting females, there was little association in profiles of hormones typically considered important for regulating heightened metabolism and nesting activity. This disassociation could arise because hormonal action may be altered in breeding female green turtles to facilitate reproductive processes.     

Orientation and behaviour of hatchling green turtles (Chelonia mydas) in the sea

To investigate the initial stage of the ‘lost-year puzzle’ of sea turtle ecology, both hatchlings from the natural nesting groung at Tortuguero, Costa Rica and hatchlings from Tortuguero eggs that had hatched in a beach on Bermuda were tracked individually after their departure from these beaches. Of the Bermuda beach most were tracked by swimmers equipped with face mask and flippers and followed by a boat. Tracking off Tortuguero was done by an observer in a following boat. The data showed that non-random departure courses were maintained even when swimming hatchlings and moved over the horizon from all fixed objects on the shore. Observations were made on swimming and diving behaviour and on predator relationships of travelling turtles. The procedures described are useful research techniques and will be used for more extensive tracking in future seasons.     

Observations on the ultrastructure of the oviduct of the Costa Rican green turtle (Chelonia mydas L.)

Unpublished investigations have shown that at the light microscope level the oviduct of the green turtle (Chelonia mydas L.) has two glandular subdivisions each of which appears to be structurally homogeneous. Histochemical studies suggest that the function of the more cranial of these is albumen secretion and that this is its sole function; in its ultrastructural features also it resembles the albumen secreting subdivision (magnum) of the avian oviduct except that it lacks the terminal mucous region to which the formation of the thick layer of egg albumen has been attributed. Layering of albumen (thick and thin layers) is not a characteristic of turtle eggs.The more caudal glandular segment presents ultrastructural features which tend to support the hypothesis that this is the site of shell-membrane formation but the presence of eggs undergoing calcareous shell formation in this subdivision shows that despite its structural homogeneity it is functionally dualistic, secreting both shell membranes and calcareous shell. In the available material ultrastructural features indicative of the latter function could not be demonstrated and further work is required in relation to calcium localization and acid phosphatase which may play a significant rôle in calcium transportation. The glands of the avian uterus in which calcareous shell deposition occurs are believed to be concerned with the addition to the albumen of plumping water. There is evidence that this addition is not a feature of turtle eggs and this may explain the absence of comparable glands in turtle.     

Biochemical indices as correlates of recent growth in juvenile green turtles (Chelonia mydas)

Nucleic acid and protein concentrations and their ratios are increasingly used as correlates of nutritional condition and growth in marine species. However, their application in studies of reptile growth has not yet been validated. The green turtle (Chelonia mydas) is an endangered marine reptile for which assessing population health requires knowledge of demographic parameters such as individual growth rates. The purpose of this study was to evaluate a number of biochemical indices ([DNA], [RNA], RNA:DNA ratio, [protein], protein:DNA ratio, and RNA:protein ratio) in liver, heart, and blood as potential predictors of recent growth rate in juvenile green turtles under controlled feeding conditions. Intake of juvenile green turtles was manipulated over twelve weeks to obtain a range of growth rates. With the exception of [RNA]_blood, [DNA]_heart, and [protein]:[DNA]_liver, all biochemical indices demonstrated significant linear relationships with growth rate during the last 1.5 weeks of the study. The best single predictors of recent growth were hepatic [RNA] and [RNA]:[protein], which explained 66% and 49%, respectively, of the variance in growth. Contrary to expectations, these two indices were negatively correlated with growth rate. To investigate the possibility that hepatic [RNA] was higher in slow-growing turtles because of elevated expression of antioxidant genes, we quantified glutathione peroxidase activity and total antioxidant potential. Both measures of antioxidant function were affected by intake and growth histories, but these effects did not explain our results for hepatic RNA and protein concentrations. We developed a model that predicted 68% of the variance in specific growth rate (SGR) with the equation SGR = − 0.913(ln[RNA]_liver) + 17.689(Condition Index) + 4.316. In addition, our findings that [DNA] and [RNA]:[DNA] for blood were significantly correlated with SGR demonstrate the potential utility of minimally invasive tissue sampling that could facilitate instantaneous population monitoring.     

Fuel use and corticosterone dynamics in hatchling green sea turtles (Chelonia mydas) during natal dispersal

During their natal dispersal hatchling sea turtles depart their nest, beach and inshore areas quickly to move into offshore developmental habitat using their finite energy stores. Patterns of fuel use and endocrine responses that could facilitate hatchling sea turtle dispersal activity are poorly understood. This study, examined aspects of intermediary metabolism by measuring plasma fuel use and an endocrine response of hatchling green turtles (Chelonia mydas) during terrestrial and aquatic activity coinciding with natal dispersal. Specifically, we measured plasma concentrations of glucose, non-esterised free fatty acids and protein to gauge the contributions of carbohydrate, lipid and protein metabolism for fuelling natal dispersal. In addition, we measured plasma levels of the steroid hormone corticosterone (CORT) a hormone implicated in regulating a number of metabolic events associated with migration and energy use in vertebrates. During terrestrial activity, hatchlings ascended through the sand from their nests and exhibited significant increases in plasma CORT and lactate indicating intense periods of anaerobic activity. During swimming, all plasma metabolites, with the exception of plasma protein, peaked between 1 and 4 h post-beginning swimming activity. Plasma CORT peaked at between 3 and 5 h of swimming activity. These plasma concentrations are consistent with intensive activity inducing catabolism of carbohydrate, lipid and protein stores to support prolonged activity. These results are similar to other vertebrates and suggest a relatively uniform cascade of physiological processes during such arduous migratory events.     

Ultrastructural features and elemental distribution in eggshell during pre and post hatching periods in the green turtle, Chelonia mydas at Ras Al-Hadd, Oman

Eggshells were randomly collected from turtle nests immediately after oviposition and at the end of incubation to examine the ultrastructural features using scanning JSM-5600LV microscopy. Three layers were recognized; an outer calcareous, a middle multistrata and an inner membrane. The calcareous layer had loose nodular units varying in shape and size without interlocking attachments. In freshly laid eggs, each nodular unit had spicules arranged in folded stacks. The spicules became unfolded during incubation, to form radiating configurations. Elemental composition and mapping of the layers were analyzed using energy dispersive spectroscopy (EDS). The elements were unevenly distributed throughout the eggshell and Ca²⁺ decreased significantly after hatching. X-ray diffraction was used to identify the crystals of the eggshells. It revealed that nodular units of the calcareous were made up of CaCO₃, as aragonite (91%), calcite (6%) and vaterite (3%). The middle layer consisted of organic amorphous material with aragonite (89%) and calcite (11%). The shell membrane consisted of reticular fibers with crystals predominantly of NaCl halite. Thermogravimetry analysis of the calcareous layer indicated a complete evaporation of bonded H₂O at 480 °C and CO₂ at 830 °C. Using the differential thermal analysis (DTA), aragonite was transformed to stable calcite at 425 °C.     

Multiple paternity assessed using microsatellite markers, in green turtles Chelonia mydas (Linnaeus, 1758) of Ascension Island, South Atlantic

Paternity was determined for three clutches and up to 20 offspring per clutch in the green turtle (Chelonia mydas) from Ascension Island, South Atlantic, using microsatellite markers. All three clutches were sired by at least two different males. The results were compared with those of previous studies of multiple paternity in turtles. No significant difference among studies was observed in the mean contribution of the males siring the largest proportion of progeny per clutch. The present study also provides evidence for segregation distortion (meiotic drive) in turtles.     

Copillaria hepatica: Fine structure of egg shell

The structure of the Capillaria hepatica egg shell was studied with the electron microscope and correlated with light microscope histochemical observations. The shell is composed of fibrous and nonfibrous components, both of which stain for protein. The fibrous component, the major portion of the shell, consists of submicroscopic fibers. The nonfibrous component is located in the outer region of the shell but is not always visible; when present it has a reticulated appearance in electron micrographs. The fibrous component is divided into outer and inner regions. The outer region is composed of radially arranged pillars which are connected at their outer surface by a beam-like network and are anchored at the base to a compact inner region. The inner region consists of a series of concentrically arranged lamellae above which is located a nonlaminated region where the pillar bases originate. At each polar end of the shell is a single opening plugged with a material which contains acid mucopolysaccharide. The fine structure of the body of the plug is unresolvable with the electron microscope; its outer surface is impregnated with electron dense particles. Externally the shell is covered by a 250 Å thick continuous membrane which is in close opposition to the surrounding host tissue.     

Calcite and aragonite seas and the de novo acquisition of carbonate skeletons

A longstanding question in paleontology has been the influence of calcite and aragonite seas on the evolution of carbonate skeletons. An earlier study based on 21 taxa that evolved skeletons during the Ediacaran through Ordovician suggested that carbonate skeletal mineralogy is determined by seawater chemistry at the time skeletons first evolve in a clade. Here I test this hypothesis using an expanded dataset comprising 40 well‐defined animal taxa that evolved skeletons de novo in the last 600 Myr. Of the 37 taxa whose mineralogy is known with some confidence, 25 acquired mineralogies that matched seawater chemistry of the time, whereas only two taxa acquired non‐matching mineralogies. (Ten appeared during times when seawater chemistry is not well constrained.) The results suggest that calcite and aragonite seas do have a strong influence on carbonate skeletal mineralogy, however, this appears to be true only at the time mineralized skeletons first evolve. Few taxa switch mineralogies (from calcite to aragonite or vice versa) despite subsequent changes in seawater chemistry, and those that do switch do not appear to do so in response to changing aragonite–calcite seas. This suggests that there may be evolutionary constraints on skeletal mineralogy, and that although there may be increased costs associated with producing a mineralogy not favored by seawater, the costs of switching mineralogies are even greater.     

Calcite precipitation and dissolution of biogenic aragonite in shallow Ordovician calcite seas

The Ordovician was a time of extensive and pervasive low-magnesium calcite (LMC) precipitation on shallow marine sea floors. The evidence comes from field study (extensive hardgrounds and other early cementation fabrics in shallow-water carbonate sequences) and petrography (large volumes of marine calcite cement in grainstones). Contemporaneous sea-floor events, particularly relationships with boring and encrusting organisms and reworking in sequences of intraformational conglomerates, confirm the early timing of such LMC cementation, and also of widespread associated aragonite dissolution. Local evidence points to the dissolved aragonite as a significant source of the calcite cement. This scenario, and the fabrics that provide the evidence for it, are likely to be pointers to other times in the stratigraphic record when LMC was the predominant shallow marine precipitate (Calcite Sea times). The combination of rapid calcite precipitation and aragonite dissolution at a time early in the Phanerozoic when many major invertebrate groups were becoming established may have acted as an influence on the evolution of both their skeletal mineralogy and their ecology.     

Calcite and aragonite distributions in the skeletons of bimineralic bryozoans as revealed by Raman spectroscopy

Abstract. Bryozoans are among a diverse range of invertebrates capable of secreting calcium carbonate skeletons. Relatively little is known about biomineralization in bryozoans, despite the importance of understanding biomineralization processes for nanotechnology and the threats imposed by ocean acidification on organisms having calcareous skeletons. Ten species of cheilostome bryozoans that are reported to have bimineralic skeletons of calcite and aragonite are studied here using Raman spectroscopy. This technique allowed identification of the two mineral phases at submicron spatial resolution, allowing the distributions of calcite and aragonite within bryozoan skeletons to be determined with unprecedented precision. Confirming previous findings based on the use of chemical stains, most of the bimineralic species analyzed exhibited a calcitic skeletal framework, composed of basal, vertical, and inner frontal walls, having aragonite deposited subsequently onto the outer surfaces of the frontal walls. In one species ( Odontionella cyclops ), aragonite formed the superstructure above the autozooids, and in two others, traces of aragonite were detected on the undersides of the frontal shields. Using Raman spectroscopy, it was possible for the first time to determine the mineralogy of small-scale structures, including orificial rims, condyles and hinge teeth, avicularian pivotal bars and rostra, and ascopore rims and sieve plates. Even when surrounded by aragonitic frontal shields, these structures were found typically to be calcitic, the two exceptions being the aragonitic avicularia of Stylopoma inchoans and O. cyclops . Unexpectedly, the first-formed part of the basal wall at the distalmost growing edge of Pentapora foliacea was found to consist mainly of aragonite. This may point to a precursory phase of biomineralization comparable with the unusual mineralogies identified previously in the earliest-formed skeletons of members of some other invertebrate phyla.     

Early changes in the shell form of Balanus balanoides (L.)

The shell shape of isolated growing individuals of Balanus balanoides (L.) changes considerably during early growth but by the time it reaches 3 mm in length its shape is almost the same as that of the adult, and thereafter it grows uniformly in all dimensions. The early metamorphosed barnacle is extremely squat with nearly vertical parietes and a relatively large aperture. The base, initially elliptical, becomes more circular and later develops ribbed borders. The carinolateral compartments are absent in the newly metamorphosed barnacle; their development and expansion is probably the main factor in the change in shape of the base. The apex of the carina, initially the lowest, becomes the highest point of the shell. The apex of the rostrum becomes relatively depressed during growth. The angle between the parietes and the base lies between 60° and 80° in newly metamorphosed individuals and drops to 35–45° in the adult.We suggest that the significance of these changes, if considered in conjunction with the orientation to current, is to maximize adhesion and minimize the drag of water currents on the shell, and to direct the water flow into the cirral net.     

Capillaria hepatica: Relation of structure and composition of egg shell to antigen release

Clean, nonembryonated Capillaria hepatica eggs recovered from infected liver tissue by physical methods as well as eggs obtained after passage through the mouse gastrointestinal tract were examined with the electron microscope. In eggs collected by physical methods the outer membrane showed defects where it was lacking at various points or disrupted. The pillars of the outer shell were frequently broken or fractured resulting in virtual collapse or separation of the outer shell from the inner shell. No shell matrix was observed in any eggs examined. Even more dramatic effects were observed in eggs recovered following passage through the mouse gastrointestinal tract. Clean, nonembryonated eggs collected by physical methods were suspended at 5 C in phosphate-buffered saline. A large amount of protein was released initially into the medium; the amount released then fell to a low level at which it remained for several weeks. Gel diffusion tests with concentrated protein supernatant and C. hepatica egg-derived antigen were compared using appropriate antisera. Bands of identity were present in both; however, egg antigen contained other proteins not present in egg supernatant. These studies indicate that during physical collection of C. hepatica eggs, sufficient damage occurs to allow for the release of materials into host tissue during experimental egg granuloma formation. An hypothesis is presented concerning the viability of C. hepatica eggs in host liver tissue following host cellular response and the possible modes of action which trigger development of eggs after release from infected liver.     

Disturbances in the development of the egg chambers of Galleria mellonella L. (Lepidoptera) after reserpine administration

Reserpine causes some changes in the morphology of developing egg chambers when administered to the last (7th)-instar larvae of Galleria mellonella. Among the alterations, there is a numerical change in the number of egg chambers produced in one ovariole. The number of previtellogenic and postvitellogenic egg chambers was decreased significantly. Histological study revealed a series of pathological alterations in the follicular epithelium, oöcytes and trophocytes. Atrophy or proliferation of follicular cells was often observed. Strong vacuolation of the cytoplasm of cells forming the egg chambers appeared in all developmental stages, mainly at the vitellogenic stage. Changes also occurred in the surface of the ovariole tube.The observed disturbances in the developing ovarioles of the wax moth are probably the consequence of reserpine action on the neuroendocrine system.