Skeletal elements within teleost eyes and a discussion of their homology

Scleral ossicles and scleral cartilages form part of the craniofacial skeleton of many vertebrates. Some vertebrates, including all birds and most reptiles, but excluding most mammals, have scleral cartilages as well as scleral ossicles supporting their eyes. The teleost equivalent of these elements has received little attention in the literature. From radiographic and whole-mount analyses of over 400 individuals from 376 teleost species, we conclude that the teleost scleral skeletal elements (ossicles and cartilage) differ significantly from those of reptiles (including birds). Scleral ossicles in teleosts have different developmental origins, different positions within the eyeball, and different relationships with the scleral cartilaginous element than those in reptiles. From whole-mount staining of a growth series of four species of teleost (Danio rerio, Salmo salar, Esox lucius, and Alosa pseudoharengus), we interpret the development of these elements and show that they arise from within an Alcian blue-staining cartilaginous ring that develops around the eye earlier in development. We present possible scenarios on the evolution of these scleral skeletal elements from a common gnathostome ancestor, and consider that teleost scleral skeletal elements may not be homologous to those in reptiles. Our study indicates that homology cannot be assumed for these elements, despite the fact that they share the same name, scleral ossicles.     

Development of chick embryo conjunctival papillae and scleral ossicles after hydrocortisone treatment

The effects of hydrocortisone injection on the development of conjunctival papillae and scleral ossicles were studied in New Hampshire Red chick embryos.Hydrocortisone injected at several different incubation stages reduced the number of conjunctival papillae and the eventual number of scleral ossicles which were produced. Repeated injections given at selected stages caused characteristic patterns of papilla deficiency.The ability to manipulate this system predictably may prove useful in the study of cell activity and supercellular organization during the initiation of organ formation.     

Studies on skeleton formation in reptiles: Patterns of ossification in the skeleton of Chelydra serpentina (Reptilia, Testudines)

Patterns and sequence of ossification are described throughout the skeleton of Chelydra serpentina Linnaeus. Evidence is adduced documenting the decoupling of ossification processes from sequence and patterns of chondrification. Convergence of ontogenetic repatterning in the ossification of the axial skeleton in Chelydra and Squamata is discussed, as are problems of adaptive modification of ossification patterns. The development of a carapace may be correlated with changes of ossification patterns in the postcranial axial skeleton of turtles, but the most striking evidence for the adaptive modification of ossification sequence obtains from a comparison of the limb skeleton and its ossification in Chelydra and in sea turtles     

Wetness of the nest environment influences cardiac development in pre- and post-natal snapping turtles (Chelydra serpentina)

We dissected hearts from near-term embryos and hatchlings of common snapping turtles (Chelydridae: Chelydra serpentina) whose eggs had incubated on wet or dry substrates, and then dried and individually weighed the heart and yolk-free carcass from each animal. Hearts and carcasses of prenatal and neonatal animals grew at different rates, and the patterns of growth by both heart and carcass differed between wet and dry environments. Hearts grew faster, both in actual mass and in mass adjusted for variation in body size, in embryos and hatchlings whose eggs were incubated on dry substrates than in animals whose eggs were held on wet media. This finding is consistent with a hypothesis that embryos incubating in dry settings experience hypovolemia secondary to dehydration and that enlargement of the heart compensates, in part, for the associated increase in viscosity of the blood. Embryonic turtles seemingly exhibit the same plasticity and response that would be expected from other vertebrate ectotherms subjected to the physiological challenges associated with desiccation and an associated reduction in blood volume.     

BMP and Hedgehog signaling during the development of scleral ossicles

Bone development is a complex process, involving multiple tissues and hierarchical inductive interactions. The study of skeletal development has largely focused on endochondral bones while intramembranous bones, such as the scleral ossicles within the avian eye, have received less attention. Our previous research directly demonstrated the involvement of sonic hedgehog and suggested the involvement of bmp2 and 4 during the development of scleral ossicles. The bones of the sclerotic ring are induced by overlying conjunctival papillae at HH 35 and 36. Here, we examine the spatial and temporal expression patterns of ptc1, ihh, bmp2, bmp4 and bmp7. We show that the cells of conjunctival papillae express ptc1, ihh and bmp2 at these stages; coincident with shh expression previously described. Interestingly, both ihh and ptc1 are also expressed in the mesenchyme underlying the papillae unlike shh and bmp2. Bmp4 and bmp7 are not expressed in these regions at any stages examined. Furthermore, using Noggin soaked beads implanted adjacent to papillae, we provide direct evidence that the BMP family of genes are important factors in the development of scleral ossicles. Localized inhibition of BMPs in this way causes a reduced expression of ihh in the surrounding tissue demonstrating that the BMP and Hedgehog pathways interact. Our data also demonstrates that the sclerotic ring has an intrinsic ability to compensate for missing elements. The scleral ossicle system provides a unique opportunity to investigate the epithelial-mesenchymal induction of intramembranous bones of the vertebrate skull.     

Transmission of Haemogregarina balli from painted turtles to snapping turtles through the leech Placobdella ornata.

Six leeches (Placobdella ornata) were allowed to feed on a painted turtle (Chrysemys picta marginata) infected with Haemogregarina balli and subjected to a period of diapause before being allowed to feed on 2 laboratory-reared snapping turtles (Chelydra serpentina). Weekly examination of thin blood films revealed infections of the turtles at 130 days postfeeding. These observations provide support for broad host specificity of hemogregarine parasites of chelonians.     

Survival and physiological responses of hatchling blanding's turtles (Emydoidea blandingii) to submergence in normoxic and hypoxic water under simulated winter conditions

Overwintering habits of hatchling Blanding's turtles (Emydoidea blandingii) are unknown. To determine whether these turtles are able to survive winter in aquatic habitats, we submerged hatchlings in normoxic (155 mmHg Po2) and hypoxic (6 mmHg Po2) water at 4 degrees C, recording survival times and measuring changes in key physiological variables. For comparison, we simultaneously studied hatchling softshell (Apalone spinifera) and snapping (Chelydra serpentina) turtles, which are known to overwinter in aquatic habitats. In normoxic water, C. serpentina and A. spinifera survived to the termination of the experiment (76 and 77 d, respectively). Approximately one-third of the E. blandingii died during 75 d of normoxic submergence, but the cause of mortality was unclear. In hypoxic water, average survival times were 6 d for A. spinifera, 13 d for E. blandingii, and 19 d for C. serpentina. Mortality during hypoxic submergence was probably caused by metabolic acidosis, which resulted from accumulated lactate. Unlike the case with adult turtles, our hatchlings did not increase plasma calcium and magnesium, nor did they sequester lactate within the shell. Our results suggest that hatchling E. blandingii are not particularly well suited to hibernation in hypoxic aquatic habitats.     

A scanning electron microscopic study of the developing epithelial scleral papillae in the eye of the embryonic chick

Eyes of early embryonic chicks possess 14 scleral papillae, derived from the conjuctival epithelium and present as transient structures between seven and 11 days of incubation. These papillae induce the formation of the 14 scleral ossicles, which develop in the adjacent, neural crest-derived ectomesenchyme. Each papilla undergoes a predictable series of developmental changes, divided by Murrary ('43) into six morphological stages (M stages 1–6). We have confirmed his staging, and provide a scanning electron microscopic (SEM) evaluation of papilla development. The earliest stage that can be visualized with the S.E.M. is M stage 2. We describe the initial modifications of the surface of papilla cells, the presence of large microvilli and the asymmetrical morphogenesis and growth of the papillae. Papillae are shed by a mechanism that involves elongation of the cells at the base of the papilla. Such moribund papillae consist of necrotic cells coated with fibers.     

Developmental and morphological variation in the teleost craniofacial skeleton reveals an unusual mode of ossification

We investigated the morphology and development of the scleral ossicles within the eyes of three species from three basal teleost orders, namely, the alewife (Alosa pseudoharengus; Clupeiformes), the surface morph of the Mexican tetra (Astyanax mexicanus; Characiformes) and zebrafish (Danio rerio; Cypriniformes). Two morphologies, circular and elongated, and one variation, fused elements, were identified. Zebrafish have small circular ossicles, whereas the alewife and the Mexican tetra have elongated ossicles. Surprisingly in the Mexican tetra these elements fuse at one end forming a continuous element with an antero-ventral opening; this may be typical for the Order Characiformes. Regardless of morphology, the ossicles develop via unilateral perichondral ossification of the scleral cartilage from two centers opposite one another in the eye. This unilateral type of ossification, in which only the perichondrium furthest from the retina contributes to the ossicles, has not previously been reported in any vertebrate. Because either the perichondrium and/or an extension of the perichondrium can transform into the scleral ossicle, we refer to the transitional tissue as periskeletal. Although the functional significance of the different shaped ossicles is unclear, skeletal muscle attaches directly to these bones, implying voluntary control. The morphological and developmental variation of teleost scleral ossicles makes them an ideal system for determining the genetic basis underlying phenotypic variation as well as for studying mechanisms underlying osteogenic and chondrogenic processes in teleosts. These data support our previous finding that scleral ossicles in teleosts may not be homologous to those in other vertebrates, such as reptiles.     

Eimeria filamentifera sp. n. from the Snapping Turtle, Chelydra serpentina (Linné), in Iowa

SYNOPSIS. Oocysts of Eimeria filamentifera sp. n. were isolated from 2 of 2 snapping turtles, Chelydra serpentina (Linné), in Iowa. The ovoid to ellipsoid oocysts of this species averaged 23.2 × 18.6 μm and had a length/width ratio ranging from 1.1 to 1.6. The ellipsoid sporocysts averaged 14.0 × 7.7 μm and had papillose, filament-bearing Stieda bodies. Oocyst and sporocyst residua were present, but micropyle and polar granule were not. This appears to be the 2nd species of Eimeria reported from snapping turtles, with E. chelydrae being the first.     

High-resolution analysis of salmonellae from turtles within a headwater spring ecosystem

Sediments and water from the pristine headwaters of the San Marcos River, Texas, USA, as well as swabs from biofilms on the carapace and from the cloacae of 17 musk turtles (Sternotherus odoratus) and one snapping turtle (Chelydra serpentina serpentina) caught at the same site, were analysed for salmonellae by culture and molecular techniques. Whereas enrichment cultures from sediment and water samples were negative for salmonellae in PCR- and in situ hybridization-based analyses, both techniques detected salmonellae after enrichments from both carapace and cloacae of nine (i.e. of 53%) musk turtles. Further characterization of 10 isolates obtained from the enrichment cultures of four selected individuals and confirmed as salmonellae by PCR analysis was achieved by fingerprinting techniques (rep-PCR). The results show differences between individuals and, in one case, variation among isolates from a single individual. All isolates from two individuals displayed identical profiles. These profiles were different from those obtained from the isolates of the third individual, which were, themselves, also identical for all isolates. Salmonellae were much more diverse in samples from the carapace of the last individual with five different rep-PCR profiles retrieved. Serotyping of seven isolates representative for each rep-PCR profile identified all isolates as representing Salmonella enterica subspecies enterica serotype Rubislaw, which demonstrates the presence of different strains of potentially human pathogenic salmonellae naturally occurring on turtles even within pristine environments. The frequent detection of these organisms in biofilms on the carapace opens the door for speculations on the role of this habitat as a reservoir for salmonellae, and on potential implications for turtles acting as a dispersal vector.     

The skeleton of the eye: II. Overlap of the scleral ossicles of the domestic fowl

The scleral ossicles, a ring of overlapping membrane bones, lie just outside the corneal margin in the eyes of domestic fowl. Eighty percent of the bony rings contain 14 bones; less than 1% have 13 bones; 19% have 15 bones; about 1% have 16 bones. Each bone is foreshadowed during development by a transient papilliform thickening in the overlying conjunctival epithelium. These conjunctival papillae appear on the eighth day of incubation and disappear on the twelfth day, when the corresponding preosseous membranes begin to ossify. Observations, and experiments involving the removal of specific papillae early or late in their maturation (in order to delete, or to reduce the size of, individual bones), demonstrated the following constraints on the morphogenesis of the scleral ossicular ring. (1) The number of ossicles is a function, not only of the number of papillae, but also of the distance between adjacent papillae; when two papillae lie close together, a single ossicle may arise beneath the pair. (2) There are three regions in the ring: nasal, dorsal (in both of which the bones overlap in one direction) and temporal (in which the bones overlap in the opposite direction). (3) The determinants of the direction of overlap between adjacent bones are extrinsic to the ossicles themselves and are distributed throughout each region, rather than confined to the discrete locations within each region where overlap normally occurs. (4) The three places in the ring where these regions meet are characterized by the loss (in 13-membered rings) or the addition (in 15- and 16-membered rings) of papillae and of their corresponding bones, and by transitions in the direction of overlap between the bones.     

Lymphoid aggregates in gonads of embryos, hatchlings, and young of turtles with temperature-dependent sex determination

Cellular infiltrations forming lymphoid-like aggregates were previously observed in gonads of two turtle species exhibiting temperature-dependent sex determination (TSD): at hatching in Chelydra serpentina; at and after hatching in Emys orbicularis. We show here that such aggregates are also present in gonads of Testudo graeca by the end of embryonic development, suggesting that their occurrence is general in turtles. Since in C. serpentina, infiltrations were observed mainly in testes exhibiting remnants of the germinal epithelium, it was assumed that their occurrence was an expression of maleness leading to rejection of this epithelium. The generality of this hypothesis was tested in E. orbicularis by looking for lymphoid-like aggregates in three types of gonads (testes, ovotestes, and ovaries) and for the stages at which they occur. Gonads were from embryos, hatchlings, and young incubated at various temperatures. Ovotestes obtained by treatment with an aromatase inhibitor of eggs incubated at female-producing temperature were also examined. In these gonads, the differentiation of Sertoli cells in testicular cords/tubes was ascertained by expression of SOX9. Moreover, the cell composition of aggregates was determined on electron micrographs. Aggregates appear in ovaries and ovotestes by the end of embryonic development and are present in the majority of these gonads at hatching, and at least up to one year after hatching. They are composed mainly of lymphocytes and fibroblasts. Aggregates are not present in typical testes. Since they occur in most ovaries, they cannot be seen as an expression of maleness. Rather, lymphocytic infiltration and formation of lymphoid aggregates in turtle gonads can be seen as components of the immune system, and can be under the control of gonadal endogenous sex steroids.     

Naturally abundant basophils in the snapping turtle, Chelydra serpentina, possess cytophilic surface antibody with reaginic function

Basophils constitute 50 to 63% of the blood leukocytes in Chelydra serpentina, the snapping turtle. Immunoglobulin (Ig) on the surface of the turtle basophil was detected by indirect immunofluorescence by using an IgG fraction from rabbit anti-turtle Ig serum (RATIg) and a fluoresceinated goat anti-rabbit antibody incubated at 4 degrees C. However, when the cells were incubated with RATIg at 22 degrees C, the basophil number, as determined by Wright's stain and neutral red counts, decreased dramatically. This morphologic evidence of degranulation was directly proportional to the antiserum concentration. Degranulation also correlated with cell histamine release (r = 0.73). In other experiments, turtle basophils were found to express antigen-specific surface Ig after immunization with sheep red blood cells (SRBC). Washed basophils from immunized turtles formed basophil-SRBC rosettes in vitro. Basophils from control turtles did not. Basophil-SRBC rosettes could also be induced by in vitro passive sensitization by preincubation of normal turtle basophils in the SRBC immune turtle sera. This study shows clearly that the turtle basophil has an immune capacity analogous to the mammalian basophil/mast cell. This study also contains the first direct evidence for the existence of reaginic antibody (or antibodies) in an ectothermic vertebrate. Finally, C. serpentina is proposed as a unique animal model for the study of basophil function.     

Complex Evolutionary and Genetic Patterns Characterize the Loss of Scleral Ossification in the Blind Cavefish Astyanax mexicanus

The sclera is the tough outer covering of the eye that provides structural support and helps maintain intraocular pressure. In some fishes, reptiles, and birds, the sclera is reinforced with an additional ring of hyaline cartilage or bone that forms from scleral ossicles. Currently, the evolutionary and genetic basis of scleral ossification is poorly understood, especially in teleost fishes. We assessed scleral ossification among several groups of the Mexican tetra (Astyanax mexicanus), which exhibit both an eyed and eyeless morph. Although eyed Astyanax surface fish have bony sclera similar to other teleosts, the ossicles of blind Astyanax cavefish generally do not form. We first sampled cavefish from multiple independent populations and used ancestral character state reconstructions to determine how many times scleral ossification has been lost. We then confirmed these results by assessing complementation of scleral ossification among the F₁ hybrid progeny of two cavefish populations. Finally, we quantified the number of scleral ossicles present among the F₂ hybrid progeny of a cross between surface fish and cavefish, and used this information to identify quantitative trait loci (QTL) responsible for this trait. Our results indicate that the loss of scleral ossification is common–but not ubiquitous–among Astyanax cavefish, and that this trait has been convergently lost at least three times. The presence of wild-type, ossified sclera among the F₁ hybrid progeny of a cross between different cavefish populations confirms the convergent evolution of this trait. However, a strongly skewed distribution of scleral ossicles found among surface fish x cavefish F₂ hybrids suggests that scleral ossification is a threshold trait with a complex genetic basis. Quantitative genetic mapping identified a single QTL for scleral ossification on Astyanax linkage group 1. We estimate that the threshold for this trait is likely determined by at least three genetic factors which may control the severity and onset of lens degeneration in cavefishes. We conclude that complex evolutionary and genetic patterns underlie the loss of scleral ossification in Astyanax cavefish.     

Can natural phenotypic variances be estimated reliably under homogeneous laboratory conditions?

The phenotypic variance is assumed to be greater in a more heterogeneous environment. The validity of this assumption is important for microevolutionists to extrapolate results from the laboratory to field environments. We subjected clutches of eggs from common snapping turtles (Chelydra serpentina) to a split-family design to evaluate the variability in incubation time and four size traits of neonates from eggs incubated in the laboratory and those left in situ. Mean size measurements were similar between the laboratory and the field, but incubation time was systematically longer in the field. We found no tendency among clutches for hatchlings resulting from eggs incubated in laboratory or field environments to demonstrate greater variability. Also contrary to expectation, clutches that experienced greater thermal variation in the field did not exhibit greater variation in phenotypic traits. Consequently, extrapolating results from the laboratory to the field may not always be problematic for microevolutionary analyses.