Biochemical properties of abnormal embryos of echinoderms and amphibians

Teratogen-induced abnormal embryos have been studied for more than three-quarters of a century, and the recent elucidation of some of the mechanisms of normal echinoderm and amphibian development has enabled the biochemistry of such abnormal embryos to be compared with that of normal embryos. Since abnormal embryos, in which the normal balance of cell populations is altered, can be produced easily and in large numbers, they form a useful model for the analysis of early differentiation.     

Does simultaneous UV-B exposure enhance the lethal and sub-lethal effects of aquatic hypoxia on developing anuran embryos and larvae?

Recent catastrophic global amphibian declines have been partially linked to increases in UV-B radiation as a consequence of stratospheric ozone depletion. Previous studies have shown that in the presence of other environmental stressors including aquatic pH and temperature and the presence of contaminants or pathogens, the lethal effects of UV-B on amphibian larvae are enhanced due to interactions between the stressors. Little is known about the interactions between UV-B and aquatic hypoxia, a common and significant natural stressor of amphibian larvae. We examined the potential effects of UV-B and aquatic hypoxia in combination on embryonic survival, developmental rate, body mass and locomotor performance of embryos and larvae of the striped marsh frog, Limnodynastes peronii. We found that while both UV-B and hypoxia independently had substantial negative effects on the developing embryos of L. peronii, they did not interact in a multiplicative or antagonistic manner. The effects of the stressors in combination were as might be predicted based on the knowledge of their independent actions alone (i.e. an additive effect). In all cases developing embryos exposed to both UV-B and hypoxia were more severely affected than those exposed to either UV-B or hypoxia alone. The results of this study show the importance of examining both the direct actions of individual stressors and how these may be influenced by the presence of other environmental factors.     

Carry-over effects of ultraviolet-B radiation on larval fitness in Rana temporaria

A number of studies have failed to find evidence for negative effects of ultraviolet-B radiation (UVBR) on amphibian early-embryonic performance, leading to the conclusions, first, that the embryonic stages of many species are tolerant to UVBR, and second, that the increased amount of UVBR reaching the Earth's surface is not likely to have any direct negative effects on many amphibian populations. However, possible carry-over effects of exposure to UVBR in the embryonic stages to the larval stages have received less attention. We studied the effects of UVBR experienced during the embryonic stages (age less than 11 days) on the later performance (age 11-75 days) of common frog, Rana temporaria, larvae. In a factorial laboratory experiment, newly fertilized embryos were divided into three different UVBR treatments (no UVBR (control), 1.25 kJm(-2) (normal) and 1.58 kJm(-2) (26% enhanced)), after which the individual larvae were raised until metamorphosis in the absence of UVBR. No effects of UVBR on embryonic survival rates, frequency of developmental anomalies or hatchling size were found, corroborating the earlier results indicating that R. temporaria embryos are tolerant to UVBR. However, analyses of larval performance revealed that larvae exposed to enhanced levels of UVBR as embryos suffered from an increased frequency of developmental anomalies and metamorphosed later and at a smaller size than larvae that had been protected from UVBR as embryos. These results suggest, in contrast to the earlier studies, that UVBR has direct negative effects on R. temporaria embryos, but these effects are expressed mostly or only during the later life stages. To this end, our results support the contention that carry-over effects from one life stage to another may be an important source of phenotypic variation in fitness.     

Predation byOligotricha striata caddis larvae on amphibian eggs: Effects of a high quality food on growth rate

In the field and also in the laboratoryOligotricha striata trichopteran larvae were observed to feed onRana temporaria eggs. Laboratory experiments revealed thatO. striata larvae preferred amphibian eggs to plant matter. The jelly-like sheath of the eggs was easily penetrated. A larva consumed 1 to 2 eggs per day. The supply of high quality food in the form of amphibian eggs was found to accelerate the growth ofO. striata larvae.     

Ultraviolet radiation, toxic chemicals and amphibian population declines

Abstract. As part of an overall ‘biodiversity crisis’, many amphibian populations are in decline throughout the world. Numerous factors have contributed to these declines, including habitat destruction, pathogens, increasing ultraviolet (UV) radiation, introduced non‐native species and contaminants. In this paper we review the contribution of increasing UV radiation and environmental contamination to the global decline of amphibian populations. Both UV radiation and environmental contaminants can affect amphibians at all life stages. Exposure to UV radiation and to certain contaminants can kill amphibians and induce sublethal affects in embryos, larvae and adults. Moreover, UV radiation and contaminants may interact with one another synergistically. Synergistic interactions of UV radiation with contaminants can enhance the detrimental effects of the contaminant and UV radiation.     

EFFECTS OF TWO PROTEASE INHIBITORS ON SEA URCHIN AND AMPHIBIAN EGG DEVELOPMENT*

After a review of our present knowledge about the effects of proteases and protease inhibitors on cell growth and egg fertilization, the results of experiments where sea urchin and amphibian eggs were treated with two protease inhibitors (TPCK, TLCK) are described. Cleavage was hardly affected, but gastrulation quickly stopped or was incomplete. The morphogenetic abnormalities which follow can be explained by abnormal gastrulation and other factors: persistence of remnants of the fertilization membrane in sea urchin larvae and dissociation of the ectoderm cells in Xenopus embryos.     

Amphibian metamorphosis as a model for studying the developmental actions of thyroid hormone

The thyroid hormones L-thyroxine and triiodo-Lthyronine have profound effects on postenbryonic development of most vertebrates.Analysis of their action in mammals is vitiated by the exposure of the developing foetus to a number of maternal factors which do not allow one to specifically define the role of thyroid hormone (TH) or that of other hormones and factors that modulate its action.Amphibian metamorphosis is obligatorily dependent on TH which can initiate all the diverse physiological manifestations of this postembryonic developmental process(morphogenesis,cell death,re-structuring,etc.) in free-living embryos and larvas of most anurans.This article will first describe the salient features of metamorphosis and its control by TH and other hormones.Emphasis will be laid on the key role played by TH receptor (TR),in particular the phenomenon of TR gene autoinduction,in initiating the developmental action of TH.Finally,it will be argued that the findings on the control of amphibian metamorphosis enhance our understanding of the regulation of postembryonic development by TH in other vertebrate species.     

Amphibian metamorphosis as a model for studying the developmental actions of thyroid hormone.

The thyroid hormones L-thyroxine and triiodo-L-thyronine have profound effects on postembryonic development of most vertebrates. Analysis of their action in mammals is vitiated by the exposure of the developing foetus to a number of maternal factors which do not allow one to specifically define the role of thyroid hormone (TH) or that of other hormones and factors that modulate its action. Amphibian metamorphosis is obligatorily dependent on TH which can initiate all the diverse physiological manifestations of this postembryonic developmental process (morphogenesis, cell death, re-structuring, etc.) in free-living embryos and larvae of most anurans. This article will first describe the salient features of metamorphosis and its control by TH and other hormones. Emphasis will be laid on the key role played by TH receptor (TR), in particular the phenomenon of TR gene autoinduction, in initiating the developmental action of TH. Finally, it will be argued that the findings on the control of amphibian metamorphosis enhance our understanding of the regulation of postembryonic development by TH in other vertebrate species.     

Sublethal Effects of Nitrite on Eastern Tiger Salamander (<Emphasis Type="Italic">Ambystoma tigrinum tigrinum</Emphasis>) and Wood Frog (<Emphasis Type="Italic">Rana sylvatica</Emphasis>) Embryos and Larvae: Implications for Field Populations

Ephemeral pools, which can have high animal biomass and low dissolved oxygen, may be prone to nitrite accumulation. As such, it is important to understand how exposure to nitrite might affect development and growth of amphibians that breed in these ephemeral pools. Wood frog (Rana sylvatica) and eastern tiger salamander (Ambystoma tigrinum tigrinum) embryos and tadpoles and young larvae were exposed to elevated concentrations of nitrite derived from sodium nitrite: 0, 0.3, 0.6, 1.2, 2.1, 4.6, and 6.1 mg l⁻¹ NO₂–N. Increasing nitrite exposure slowed embryonic and larval development in both the eastern tiger salamander and the wood frog, reduced growth in tiger salamander embryos and larvae, and delayed metamorphosis in the wood frog. At concentrations less than 2 mg l⁻¹ NO₂–N nitrite delayed hatching, and at concentrations above 2 mg l⁻¹ time to hatching decreased causing more individuals to hatch at less developed stages. Nitrite also increased asynchrony in tiger salamander hatching. The sublethal effects of nitrite on amphibian development, growth and hatching could have serious repercussions on amphibian fitness in ephemeral environments. Potential increases in mortality on field populations caused by sublethal effects of nitrite are discussed.     

Synergistic effects of the invasive Chinese tallow (Triadica sebifera) and climate change on aquatic amphibian survival

Changes in climate and the introduction of invasive species are two major stressors to amphibians, although little is known about the interaction between these two factors with regard to impacts on amphibians. We focused our study on an invasive tree species, the Chinese tallow (Triadica sebifera), that annually sheds its leaves and produces leaf litter that is known to negatively impact aquatic amphibian survival. The purpose of our research was to determine whether the timing of leaf fall from Chinese tallow and the timing of amphibian breeding (determined by weather) influence survival of amphibian larvae. We simulated a range of winter weather scenarios, ranging from cold to warm, by altering the relative timing of when leaf litter and amphibian larvae were introduced into aquatic mesocosms. Our results indicate that amphibian larvae survival was greatly affected by the length of time Chinese tallow leaf litter decomposes in water prior to the introduction of the larvae. Larvae in treatments simulating warm winters (early amphibian breeding) were introduced to the mesocosms early in the aquatic decomposition process of the leaf litter and had significantly lower survival compared with cold winters (late amphibian breeding), likely due to significantly lower dissolved oxygen levels. Shifts to earlier breeding phenology, linked to warming climate, have already been observed in many amphibian taxa, and with most climate models predicting a significant warming trend over the next century, the trend toward earlier breeding should continue if not increase. Our results strongly suggest that a warming climate can interact with the effects of invasive plant species, in ways we have not previously considered, to reduce the survival of an already declining group of organisms.     

Studies on the Effects of Bromodeoxyuridine (BUdR) on Differentiation

The effects of BUdR on morphogenesis in Acetabularia mediterranea and on embryonic development (sea urchin, tunicate and amphibian embryos) have been studied.
In the presence of BUdR, cap development is affected in both the anucleate and nucleate fragment of the unicellular alga Acetabularia mediterranea . In the nucleate fragment, the initiation of cap formation can be inhibited. BUdR exerts various inhibitory effects on embryonic development. Modifications of sensitivity to BUdR during development are also observed: in Paracentrotus , a change in sensitivity towards the drug occurs at the 16 cell stage, while in amphibian eggs, a modification can be shown after the gastrula stage.
The significance of these observations is discussed.     

Ciliary cells in the epidermis of the larval Australian dipnoan, Neoceratodus

Newly hatched Australian lungfish, Neoceratodus forsteri KrefFt, develop a ciliary current over the skin surface which continues for more than six weeks of larval life. The gill surface under the opercula also becomes ciliated. The ciliary cells producing the current are found to develop in like manner to those in several amphibian embryos and larvae. The long duration of ciliary currents in one of the most primitive living gnathostomes has ontogenetic and phylogenetic implications.     

Locomotion in Amphibian Larvae (or "Why Aren't Tadpoles Built Like Fishes?")

SYNOPSIS. A variety of morphological features that affect locomotiondistinguish larvae of the three living amphibian orders fromfishes and their larvae. The oddest amphibian larvae are anurantadpoles. With their globose bodies, concealed forelimbs, abruptlycompressed and terminally tapered tails, tadpoles not only differradically from fishes but they—unlike caecilians or salamanders—alsodiffer radically from their adults. Tadpoles typically haveless axial musculature and much simpler myotomes than fishes.Surprisingly, in terms of mechanical (propeller) efficiencyand maximum sprint speeds, tadpoles still perform as well asmany teleosts of comparable sizes. From a consideration of hydromechanics,no amphibian larvae appear to be designed for sustained swimmingat high speeds. High maneuverability, rather than sustainablespeed, are important for amphibian larval survival.Two key featuresof tadpoles are the absence of caudal vertebrae and unexposedpectoral appendages. With only a notochord to serve as a skeleton,the tadpole tail is extremely flexible. Because of this exceptionalflexibility, tadpoles can fold their tails up against the bodyand turn rapidly with virtually no displacement of their centerof mass. Caudal flexibility can be regulated by muscle activityin the tadpole to effect turning. Lateral appendages are notneeded for this movement and are free to develop directly intotheir adult morphology; the anterior ones develop under coverof an opercular fold where they do not contribute to drag. Acase is presented, based on the ecology of metamorphosis, thatanuran transformation should be as brief as possible. With nobone to resorb, metamorphosis of the anuran caudal appendagecan, indeed, be very rapid.The basic kinematics of constantvelocity straightforward swimming for tadpoles and salamanderlarvae is reviewed, as well as the kinematics and electromyographyof starting, stopping, and turning in tadpoles. An attempt ismade to relate swimming kinematics to the characteristic morphologiesof amphibian larvae. Swimming speed in Rana, Bufo and Aynbystomalarvae, which swim only intermittently, is modulated by changingtail beat frequency. However, Xenopus, which swims constantlyby sculling with its tail, regulates swimming speed (at lowto intermediate velocities) by varying the length of the propulsivewave in its tail. Xenopus and Rana differ in the morphologyof their notochord, spinal cord, spinal nerves, and spinal motorpool distribution within the spinal cord. These differencesmay underlie the different way these larvae regulate swimming.They may also reflect their phylogenetic history.     

The Effects of Ultraviolet Radiation on the Biology of Amphibians

SYNOPSIS. Potential causes for the global decline of amphibiansinclude habitat loss, disease, environmental contaminants andclimate changes. The diminishing ozone layer and consequentincrease of ultraviolet-B radiation reaching the earth's surfacehas been hypothesized to be a mortality factor, especially inhabitats otherwise undisturbed. We discuss the fundamental physicsof UV and types of biological damage after exposure. A deleteriouschange in DNA, especially the production of pyrimidine dimers,is a main effect of UVB exposure. Damaged DNA can be repairedby enzymes such as photolyase when organisms are irradiatedwith UVA or visible light. We review studies including laboratoryand field approaches on damage to amphibians from UVB exposure.Field studies in which embryos were exposed to natural sunlightor sunlight with UVB removed have shown conflicting results:some show increased embryonic mortality after UVB exposure,whereas others show that current levels of UVB are not detrimentalto amphibian embryos. The abiotic factors such as water depth,water color, and dissolved organic content of aquatic ovipositionsites effectively reduces UVB penetration through water andreduces exposure to UVB of all life history stages. Biotic factorssuch as jelly capsules around eggs, melanin pigmentation ofeggs, and color of larvae and metamorphosed forms further reduceeffectiveness of UVB penetration. We suggest areas of futureresearch to test the hypothesis of the causal connection betweencurrent UVB levels and amphibian decline.     

Tailoring science outreach through E-matching using a community-based participatory approach

In an effort to increase science exposure for pre-college (K-12) students and as part of the science education reform agenda, many biomedical research institutions have established university-community partnerships. Typically, these science outreach programs consist of pre-structured, generic exposure for students, with little community engagement. However, the use of a medium that is accessible to both teachers and scientists, electronic web-based matchmaking (E-matching) provides an opportunity for tailored outreach utilizing a community-based participatory approach (CBPA), which involves all stakeholders in the planning and implementation of the science outreach based on the interests of teachers/students and scientists. E-matching is a timely and urgent endeavor that provides a rapid connection for science engagement between teachers/students and experts in an effort to fill the science outreach gap. National Lab Network (formerly National Lab Day), an ongoing initiative to increase science equity and literacy, provides a model for engaging the public in science via an E-matching and hands-on learning approach. We argue that science outreach should be a dynamic endeavor that changes according to the needs of a target school. We will describe a case study of a tailored science outreach activity in which a public school that serves mostly under-represented minority students from disadvantaged backgrounds were E-matched with a university, and subsequently became equal partners in the development of the science outreach plan. In addition, we will show how global science outreach endeavors may utilize a CBPA, like E-matching, to support a pipeline to science among under-represented minority students and students from disadvantaged backgrounds. By merging the CBPA concept with a practical case example, we hope to inform science outreach practices via the lens of a tailored E-matching approach.     

Identification of morphogenetic capability limitations via a single starfish embryo/larva reconstruction method

Reconstruction of a starfish embryo provides unique morphogenesis during the developmental process that is not observed in normal development. Here, we established a novel method for reconstruction from single embryos/larvae. By using this method, we investigated the morphogenetic capabilities in critical steps during the reconstruction process as showed by the reconstructed embryos generated from embryos/larvae at the six developmental stages, or from segregated ectodermal and/or endomesodermal cells. Additionally, the novel method addressed several problems found in prior methods related to reproducibly generating reconstructed embryos. In the reconstructions from the various stage embryos/larvae, the morphogenetic capabilities were substantively reduced in the reconstructed embryos generated from 3‐day bipinnaria (3dBp). The combination experiments using ectodermal or endomesodermal cells segregated from 2dBp or 3dBp showed a reduction of the morphogenetic capabilities in both cells types in 3dBp. The reconstructed embryos generated from ectodermal or endomesodermal cells segregated from 2dBp possessed partial morphological features, such as formation of the epithelium or blastopore, but all failed to develop into bipinnariae. These results indicate two limitations of the morphogenetic capabilities during the reconstruction process. Firstly, the morphogenetic capabilities to reconstruct an embryo are considerably reduced between 2dBp and 3dBp. Secondly, cells specified as ectoderm or endomesoderm possess limited morphogenetic capabilities to reconstruct bipinnaria. Furthermore, our results demonstrate that the interaction between these specified cell types is required for reconstruction.     

The effects of nest temperature,nest substrate,and clutch size on the oxygenation of embryos and larvae of the Australian moss frog,Bryobatrachus nimbus

The jelly around amphibian eggs presents a formidable barrier to oxygen diffusion. Therefore, egg capsules must be thin enough, and the dimensions of globular egg masses small enough, to avoid oxygen limitation leading to developmental retardation or death. The eggs of the Australian moss frog, Bryobatrachus nimbus, have the thickest jelly capsule known for any anuran amphibian. Laboratory measurements of respirometric variables predict that single prehatching embryos should be normoxic between 5 degrees and 20 degrees C, with Po(2 in) maintained above critical levels (10.2-17.0 kPa). However, numerical models of embryos amid larger egg masses (13-20 eggs) predict hypoxia at temperatures above 5 degrees C. Contrary to model predictions, however, B. nimbus embryos rarely experience hypoxia in natural nests, because embryos occur in one or two layers and the moss substrate permits aeration of the lower surface while photosynthesis probably supplies oxygen directly. After hatching, larvae move to oxygen-rich regions of the jelly mass and disperse more widely within the mass as temperatures increase. Although nest characteristics relieve diffusive constraints, small clutch sizes, low rates of embryonic and larval respiration, and the cool climate occupied by B. nimbus are the main characteristics that prevent hypoxia.     

Immunocytochemistry of the amphibian embryo--from overview to ultrastructure

Amphibian embryos are standard research objects to study pattern formation and morphogenesis. Due to their external development and robust nature, experimental manipulations such as microinjections or transplantations can be easily performed. However, most immunocytochemical approaches addressing the specific localization of proteins are hampered by the fragility of the large and yolky embryonic cells which render high resolution staining difficult. Immunocytochemical data are therefore often restricted to either overall patterns in whole embryo preparations or to immunofluorescent localization with limited resolution on sections. High resolution or ultrastructural protein localization data are rare and can be achieved only with time consuming procedures. Here, a comparative study of immunocytochemical methods suitable for light and electron microscopy using different kinds of plastic resins is presented. Three main approaches are described: preembedding staining of whole embryos, postembedding staining of ultrathin sections and preembedding staining of vibratome sections. All the procedures are designed to study protein expression in early amphibian embryos en gros as well as en detail and the described techniques are suitable to combine two or three levels of resolution on the very same biological specimen. Examples are presented and advantages and disadvantages of the different protocols are discussed.     

Plasticity of hatching in amphibians: evolution, trade-offs, cues and mechanisms

Many species of frogs and salamanders, in at least 12 families, alter their timing of hatching in response to conditions affecting mortality of eggs or larvae. Some terrestrially laid or stranded embryos wait to hatch until they are submerged in water. Some embryos laid above water accelerate hatching if the eggs are dehydrating; others hatch early if flooded. Embryos can hatch early in response to predators and pathogens of eggs or delay hatching in response to predators of larvae; some species do both. The phylogenetic pattern of environmentally cued hatching suggests that similar responses have evolved convergently in multiple amphibian lineages. The use of similar cues, including hypoxia and physical disturbance, in multiple contexts suggests potential shared mechanisms underlying the capacity of embryos to respond to environmental conditions. Shifts in the timing of hatching often have clear benefits, but we know less about the trade-offs that favor plasticity, the mechanisms that enable it, and its evolutionary history. Some potentially important types of cued hatching, such as those involving embryo-parent interactions, are relatively unexplored. I discuss promising directions for research and the opportunities that the hatching of amphibians offers for integrative studies of the mechanisms, ecology and evolution of a critical transition between life-history stages.     

Paraquat-induced oxidative stress response during amphibian early embryonic development

In addition to the endogenous production of reactive oxygen species (ROS) as a result of normal development, amphibian external development often forces embryos to deal with oxidative stress-producing agents present in the environment. Embryos should therefore develop protective systems to reduce ROS toxicity and achieve successful development. The present work was aimed to characterize the effects produced by the widespread-used ROS-generator pesticide Paraquat during early embryonic development in the toad Chaunus arenarum, as well as to get insights into the defense response elicited by amphibian embryos. The approach consisted in generating a sharp and brief oxidative stress condition early during embryonic development to stimulate the cellular mechanisms involved in ROS-antioxidant response. Results revealed that Paraquat-treatment reduced the ability of embryos to develop normally, leading to arrests of development and severe malformations such as tail abnormalities, abdominal edema, reduced head development and curved dorsal structures. Although Paraquat effects were morphologically evident from gastrula stage on, alterations such as chromatin condensation were observed even at blastula stage by histological examinations. Regarding detoxifying enzymes, a significant induction of Mn-superoxide dismutase activity was detected at stages beyond gastrula in embryos surviving Paraquat treatment, suggesting a major role of this enzyme in the antioxidant response during early embryonic development.